PH-sensitive polymeric micelles for drug delivery

ABSTRACT

Mixed micelles containing poly(L-histidine)-poly(ethylene glycol) block copolymer and poly(L-lactic acid)-poly(ethylene glycol) block copolymer are a pH-sensitive drug carrier that release the drug in an acidic microenvironment, but not in the blood. Since the microenvironment of solid tumors is acidic, these mixed micelles are useful for treating cancer, including those cancers exhibiting multidrug resistance. Targeting ligands, such as folate, can also be attached to the mixed micelles for enhancing drug delivery into cells. Methods of treating a warm-blooded animal with such a drug are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/640,739, filed May 19, 2003, which claims the benefit of U.S.Provisional Application No. 60/381,970, filed May 19, 2002, both ofwhich are hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

This invention relates to drug delivery. More particularly, thisinvention relates to polymeric micelles for targeted drug delivery,including drug delivery for treating cancer and bypassing multidrugresistance of cancer cells by taking advantage of tumor and endosomalpH.

Although important findings in scientific research and technologicaladvances, such as long-circulating carriers, enhanced permeation andretention (EPR) effect, and receptor-mediated endocytosis, have beenachieved in the last decades for effective solid tumor targeting,chemotherapy still faces a major challenge for improving specific drugaccumulation in tumor sites. Z. Ning et al., Increased microvascularpermeability contributes to preferential accumulation of stealthliposomes in tumor tissue, 53 Cancer Res. 3764-3770 (1993); S. M.Moghimi et al., Long-circulating and target-specific nanoparticles:Theory and practice, 53 Pharmacol. Rev. 283-318 (2001); D. Putnam & J.Kopecek, Polymer conjugates with anticancer activity, 122 Adv. Polymer.Sci. 57-123 (1995); D.C. Drummond et al., Optimizing liposomes fordelivery of chemotherapeutic agents to solid tumors, 51 Pharmcol. Rev.691-743 (1999); M. Yokoyama, Novel passive targetable drug delivery withpolymeric micelles, in Biorelated Polymers and Gels 193-229 (T. Okanoed. 1998).

The tumor extracellular pH (pH_(e)) is a consistently distinguishingphenotype of most solid tumors from surrounding normal tissues. Themeasured pH values of most solid tumors in patients, using invasivemicroelectrodes, range from pH 5.7 to pH 7.8 with a mean value of 7.0.More than 80% of these measured values are below pH 7.2, while normalblood pH remains constant at pH 7.4. K. Engin et al., Extracellular pHdistribution in human tumors, 11 Int. J. Hyperthermia 211-216 (1995); R.van Sluis et al., In vivo imaging of extracellular pH using ¹H MRSI, 41Magn. Reson. Med. 743-750 (1999); A. S. E. Ojugo et al., Measurement ofthe extracellular pH of solid tumors in mice by magnetic resonancespectroscopy: a comparison of exogenous ¹⁹F and ³¹P probes, 12 NMRBiomed. 495-504 (1999). The acidity of tumor interstitial fluid ismainly attributed, if not entirely, to the higher rate of aerobic andanaerobic glycolysis in cancer cells (proton production by lactateformation and ATP lysis) than normal cells. M. Stubbs et al., Causes andconsequences of tumour acidity and implications for treatment, 6 Opinion15-19 (2000). Such acidic extracellular pH has prompted researchers toattempt to establish pH-sensitive anticancer drug delivery systems, suchas pH-sensitive liposomes. However, effective systems have not beenachieved because of lack of proper pH-sensitive functional groups in thephysiological pH range. O. V. Gerasimov et al., Cytosolic drug deliveryusing pH- and light-sensitive liposomes, 38 Adv. Drug Deliv. Rev.317-338 (1999); 0. Meyer et al., Copolymers of N-isopropylacrylamide cantrigger pH sensitivity to stable liposomes, 421 FEBS Lett. 61-64 (1998).Recently, water soluble polymers modified with sulfonamideself-assembled nanoparticles showed enhanced drug release, interactionwith and internalization into cells at tumor pH. K. Na & Y. H. Bae,Self-assembled hydrogel nanoparticles responsive to tumor extracellularpH from pullulan derivative/sulfonamide conjugate: Characterization,aggregation and adriamycin release in vitro, 19 Pharm. Res. 681-688(2002); S. K. Han, K. Na & Y. H. Bae, Sulfonamide based pH-sensitivepolymeric micelles: physicochemical characteristics and pH-dependentaggregation, Colloids. Surf. A. Physicochem. Eng. Aspects 001-11 (2002);K. Na, E. S. Lee & Y. H Bae, Adriamycin loaded pullulanacetate/sulfonamide conjugate nanoparticles responding to tumor pH:pH-dependent cell interaction, internalization and cytotoxicity invitro, 87 J. Contr. Release 3-13 (2003).

In view of the foregoing, it will be appreciated that providing apH-dependent drug carrier that releases a drug in the acidicmicroenvironment of solid tumors while maintaining stability in theblood would be a significant advancement in the art.

BRIEF SUMMARY OF THE INVENTION

An illustrative embodiment of the present invention comprises mixedmicelles comprising poly(L-histidine)-poly(ethylene glycol) blockcopolymer and poly(L-lactic acid)-poly(ethylene glycol) block copolymer.Illustratively, the poly(L-histidine) portion has a molecular weight ofat least about 5,000, and the poly(ethylene glycol) portion has amolecular weight of at least about 2,000. These mixed micelles comprisea hydrophobic interior portion, which can receive a drug, such as ahydrophobic drug. Included among such hydrophobic drugs are anticancerdrugs, such as adriamycin (doxorubicin). These mixed micelles arepH-sensitive in that they are stable in the microenvironment of theblood, but are unstable and release the drug in the acidicmicroenvironment of solid tumors.

The mixed micelles can comprise varying amounts of thepoly(L-histidine)-poly(ethylene glycol) block copolymer and of thepoly(L-lactic acid)-poly(ethylene glycol) block copolymer. Illustrativeembodiments of the mixed micelles comprise about 60-90% by weight ofpoly(L-histidine)-poly(ethylene glycol) block copolymer and about 10-40%by weight of poly(L-lactic acid)-poly(ethylene glycol) block copolymer.A typical embodiment within this range would comprise about 75% byweight of poly(L-histidine)-poly(ethylene glycol) block copolymer andabout 25% by weight of poly(L-lactic acid)-poly(ethylene glycol) blockcopolymer.

The poly(L-histidine)-poly(ethylene glycol) block copolymer and/or thepoly(L-lactic acid)-poly(ethylene glycol) block copolymer can alsocomprise a folate residue for targeting drug delivery by folate-receptormediated endocytosis.

Other illustrative embodiments of the present invention comprisecompositions of matter comprising poly(L-histidine)-poly(ethyleneglycol) block copolymer and poly(L-histidine)-poly(ethylene glycol)block copolymer-folate. Still other illustrative embodiments of theinvention comprise intermediates useful in the synthesis of these blockcopolymer compositions, including N^(α)-carbobenzoxy-L-histidine,N^(α)-carbobenzoxy-N^(im)-dinitrophenyl-L-histidine,N^(im)-dinitrophenyl-L-histidine N-carboxyanhydride, andpoly(N^(im)-dinitrophenyl-L-histidine).

Another illustrative embodiment of the invention comprises a method ofmaking an imidazole-protected L-histidine N-carboxyanhydride comprising:

-   -   (a) reacting L-histidine, which comprises an a-amino group and        an imidazole group, with an amino-group protecting reagent,        resulting in an a-amino-protected L-histidine;    -   (b) reacting the a-amino-protected L-histidine with an        imidazole-protecting reagent, resulting in an a-amino-protected        and imidazole-protected L-histidine; and    -   (c) reacting the a-amino-protected and imidazole-protected        L-histidine with an anhydride-forming reagent, resulting in the        imidazole-protected L-histidine N-carboxyanhydride.

In certain illustrative embodiments, the amino-group protecting reagentcomprises benzyl chloroformate and the α-amino-protected L-histidinecomprises N^(α)-carbobenzoxy-L-histidine. Further, theimidazole-protecting reagent can comprise 2,4-dinitrofluorobenzene, andthe α-amino-protected and imidazole-protected L-histidine comprisesN^(α)-carbobenzoxy-N^(im)-dinitrophenyl-L-histidine. Further, anillustrative anhydride-forming reagent comprises thionyl chloride. Stillfurther, the imidazole-protected L-histidine N-carboxyanhydride cancomprise N^(im)-dinitrophenyl-L-histidine N-carboxyanhydride.

Yet another illustrative embodiment of the invention comprises a methodof making N^(im)-dinitrophenyl-L-histidine N-carboxyanhydridecomprising:

-   -   (a) reacting L-histidine with benzyl chloroformate, resulting in        an N^(α)-carbobenzoxy-L-histidine;    -   (b) reacting the N^(α)-carbobenzoxy-L-histidine with        2,4-dinitrofluorobenzene, resulting in an        N^(α)-carbobenzoxy-N^(im)-dinitrophenyl-L-histidine; and    -   (c) reacting the        N^(α)-carbobenzoxy-N^(im)-dinitrophenyl-L-histidine with thionyl        chloride, resulting in the N^(im)-dinitrophenyl-L-histidine        N-carboxyanhydride.

Still another illustrative embodiment of the invention comprises amethod of making an imidazole-protected poly(L-histidine) comprising:

-   -   (a) reacting L-histidine, which comprises an a-amino group and        an imidazole group, with an amino-group protecting reagent,        resulting in an a-amino-protected L-histidine;    -   (b) reacting the a-amino-protected L-histidine with an        imidazole-protecting reagent, resulting in an α-amino-protected        and imidazole-protected L-histidine;    -   (c) reacting the a-amino-protected and imidazole-protected        L-histidine with an anhydride-forming reagent, resulting in an        imidazole-protected L-histidine N-carboxyanhydride; and    -   (d) converting the imidazole-protected L-histidine        N-carboxyanhydride to an acid addition salt thereof and then        polymerizing the imidazole-protected L-histidine        N-carboxyanhydride acid addition salt by ring-opening        polymerization in the presence of an initiator to result in the        imidazole-protected poly(L-histidine).

Another illustrative aspect of the invention comprises a method ofmaking poly(N^(im)-dinitrophenyl-L-histidine) comprising:

-   -   (a) reacting L-histidine with benzyl chloroformate, resulting in        N^(α)-carbobenzoxy-L-histidine;    -   (b) reacting the N^(α)-carbobenzoxy-L-histidine with        2,4-dinitrofluorobenzene, resulting in        N-carbobenzoxy-N^(im)-dinitrophenyl-L-histidine;    -   (c) reacting the N-carbobenzoxy-N^(im)-dinitrophenyl-L-histidine        with thionyl chloride, resulting in an        N^(im)-dinitrophenyl-L-histidine N-carboxyanhydride; and    -   (d) converting the N^(im)-dinitrophenyl-L-histidine        N-carboxyanhydride to N^(im)-dinitrophenyl-L-histidine        N-carboxyanhydride hydrochloride and then polymerizing the        N^(im)-dinitrophenyl-L-histidine N-carboxyanhydride        hydrochloride by ring-opening polymerization in the presence of        n-hexylamine or isopropylamine to result in the        poly(N^(im)-dinitrophenyl-L-histidine).

Yet another illustrative aspect of the invention comprises a method ofmaking poly(L-histidine) comprising:

-   -   (a) polymerizing N^(im)-dinitrophenyl-L-histidine        N-carboxyanhydride hydrochloride by ring-opening polymerization        in the presence of an initiator to result in        poly(N^(im)-dinitrophenyl-L-histidine); and    -   (b) deprotecting the poly(N^(im)-dinitrophenyl-L-histidine) to        result in poly(L-histidine).

Still another illustrative embodiment of the invention comprises amethod of making a poly(L-histidine)-poly(ethylene glycol) blockcopolymer comprising:

-   -   (a) reacting monocarboxylic acid-poly(ethylene glycol),        comprising a carboxylic acid group, with an activating reagent        to result in an activated monocarboxylic acid-poly(ethylene        glycol) wherein the carboxylic acid group is activated;    -   (b) reacting poly(N^(im)-dinitrophenyl-L-histidine), comprising        a terminal α-amino group, with the activated monocarboxylic        acid-poly(ethylene glycol) such that an amide bond is formed        between the terminal a-amino group of        poly(N^(im)-dinitrophenyl-L-histidine) and the carboxylic group        of activated monocarboxylic acid-poly(ethylene glycol),        resulting in a        poly(N^(im)-dinitrophenyl-L-histidine)-poly(ethylene glycol)        block copolymer; and    -   (c) deprotecting the        poly(N^(im)-dinitrophenyl-L-histidine)-poly(ethylene glycol)        block copolymer to result in the poly(L-histidine)-poly(ethylene        glycol) block copolymer.

In this embodiment, an illustrative activating reagent comprisesN-hydroxysuccinimide, and, in such case, the activated monocarboxylicacid-poly(ethylene glycol) comprises N-hydroxysuccinimide-poly(ethyleneglycol).

Still another illustrative embodiment of the invention comprise a methodof making poly(L-histidine)-poly(ethylene glycol) block copolymer-folatecomprising:

-   -   (a) activating folic acid, which comprises a carboxyl group, to        result in activated folic acid wherein the carboxyl group is        activated;    -   (b) reacting        poly(N^(im)-dinitrophenyl-L-histidine)-poly(ethylene glycol)        block copolymer, comprising a terminal hydroxyl group, with the        activated folic acid such that an ester bond is formed between        the terminal hydroxyl group of the        poly(N^(im)-dinitrophenyl-L-histidine)-poly(ethylene glycol)        block copolymer and the carboxyl group of the activated folic        acid, resulting in        poly(N^(im)-dinitrophenyl-L-histidine)-poly(ethylene glycol)        block copolymer-folate; and    -   (c) deprotecting the        poly(N^(im)-dinitrophenyl-L-histidine)-poly(ethylene glycol)        block copolymer-folate to result in the        poly(L-histidine)-poly(ethylene glycol) block copolymer-folate.

In such embodiment, the carboxyl group can be illustratively activatedwith N,N′-dicyclohexylcarbodiimide.

Another illustrative aspect of the invention comprises a method fortreating a warm-blooded animal with a drug comprising:

-   -   (a) mixing the drug with a pH-sensitive mixed micelle        composition comprising (i) poly(L-histidine)-poly(ethylene        glycol) block copolymer and poly(L-lactic acid)-poly(ethylene        glycol) block copolymer, (ii) poly(L-histidine)-poly(ethylene        glycol) block copolymer-folate and poly(L-lactic        acid)-poly(ethylene glycol) block copolymer, (iii)        poly(L-histidine)-poly(ethylene glycol) block copolymer and        poly(L-lactic acid)-poly(ethylene glycol) block        copolymer-folate, or (iv) poly(L-histidine)-poly(ethylene        glycol) block copolymer-folate and poly(L-lactic        acid)-poly(ethylene glycol) block copolymer-folate, to result in        drug-loaded mixed micelle composition; and    -   (b) administering the drug-loaded mixed micelle composition to        the animal such that the drug-loaded mixed micelle composition        is systemically circulated in the animal, wherein the        drug-loaded mixed micelle composition is stable in the blood and        the drug is released from the drug-loaded mixed micelle        composition in acidic microenvironments of solid tumors and        endosomes.

Yet another illustrative aspect of the invention comprises a method fortreating multidrug resistance in a warm-blooded animal comprisingadministering into the systemic circulation of the animal a drug-loadedmixed micelle composition comprising a mixture of a hydrophobicanticancer drug and pH-sensitive mixed micelles comprising (i)poly(L-histidine)-poly(ethylene glycol) block copolymer andpoly(L-lactic acid)-poly(ethylene glycol) block copolymer, (ii)poly(L-histidine)-poly(ethylene glycol) block copolymer-folate andpoly(L-lactic acid)-poly(ethylene glycol) block copolymer, (iii)poly(L-histidine)-poly(ethylene glycol) block copolymer andpoly(L-lactic acid)-poly(ethylene glycol) block copolymer-folate, or(iv) poly(L-histidine)-poly(ethylene glycol) block copolymer-folate andpoly(L-lactic acid)-poly(ethylene glycol) block copolymer-folate.

A still further illustrative embodiment of the present inventioncomprises a method for treating a warm-blooded animal with a drugcomprising:

-   -   (a) mixing the drug with a pH-sensitive mixed micelle        composition comprising (i) poly(L-histidine)-poly(ethylene        glycol) block copolymer or poly(L-histidine)-poly(ethylene        glycol) block copolymer-targeting moiety and (ii) an amphiphilic        polymer or amphiphilic polymer-targeting moiety, to result in a        drug-loaded mixed micelle composition; and    -   (b) administering the drug-loaded mixed micelle composition to        the animal such that the drug-loaded mixed micelle composition        is systemically circulated in the animal, wherein the        drug-loaded mixed micelle composition is stable in the blood and        releases the drug in the acidic microenvironments of solid        tumors and endosomes.

An illustrative targeting moiety according to the present inventioncomprises folate or any other targeting moiety known in the art.Illustrative amphiphilic polymers according to the present inventioncomprise poly(L-lactic acid)-poly(ethylene glycol) block copolymer,poly(DL-lactic-co-glycolic acid), and ABA block copolymers, such aspoly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) blockcopolymer.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows a schematic representation of synthesis of protectedL-histidine monomer according to the present invention.

FIG. 2 shows a schematic representation of synthesis ofpoly(L-histidine) (“polyHis”) according to the present invention.

FIG. 3A shows the pH profiles by acid-base titration ofpolyHis5K-b-PEG2K (●), polyHis5K (▪), and NaCl (▾). FIG. 3B shows the pHprofiles by acid-base titration of polyHis3K-b-PEG2K (●) and polyHis3K(▪).

FIG. 4 shows the results of a stability study of micelles (0.1 g/L) atpH 8.0 through the change of transmittance at 37° C. T/T_(i) is thetransmittance at a given time divided by the transmittance at zero time:polyHis5K-b-PEG2K (●) and polyHis3K-b-PEG2K (▪).

FIGS. 5A-B show the determination of the critical micelle concentration(CMC) from the fluorescence intensity (I₁) obtained after scanning(λ_(emission)=360-460 nm) at λ_(excitation)=339 nm with a pyreneconcentration of 1×10-6 M in NaOH—Na2B4O7 buffer (pH 8.0), wherein FIG.5A shows intensity as a function of wavelength for polyHis5K-b-PEG2Kconcentrations from 0.5 to 100 μg/mL, and FIG. 5B shows intensity as afunction of log polymer concentration.

FIG. 6 shows the pH effect on the CMC of polyHis5K-b-PEG2K polymericmicelles that were prepared at various pHs from pH 5.0 to pH 8.0.

FIGS. 7A-B show the particle size distribution of polyHis5K-b-PEG2Kpolymeric micelles measured by dynamic light scattering (DLS) using aZetasizer (FIG. 7A) and atomic force microscopy (AFM) (FIG. 7B).

FIG. 8 shows the relaxation rate G of polyHis5K-b-PEG2K polymericmicelles as a function of sin²(θ/2) at room temperature.

FIG. 9 shows the transmittance change of polyHis5K-b-PEG2K polymericmicelles (0.1 g/L) with pH. The polyHis5K-b-PEG2K polymeric micelleswere prepared in NaOH-Na2B4O7 buffer (pH 8.0) and exposed at each pH for24 h.

FIG. 10 shows the change of particle size distribution inpolyHis5K-b-PEG2K micelles exposed to pH 8.0 (●), pH 7.4 (▪), pH 7.2(▴), pH 6.8 (▾), pH 6.4 (♦), or pH 6.0 (0) for 24 h.

FIG. 11 shows the change of pyrene fluorescence intensity (I₁) with pHat constant micelle concentration (0.1 g/L). The polyHis5K-b-PEG2Kpolymeric micelles were prepared in NaOH (or HCl)-Na2B4O7 buffer (pH8.0) and exposed to each pH for 24 h.

FIG. 12 shows pH-dependent relative turbidity (T/T_(i)%) of mixedmicelles comprising polyHis/PEG and PLLA/PEG (PLLA/PEG content: 0 wt %(∘); 5 wt % (●); 10 wt % (▪); 25 wt % (▴); 40 wt % (▾); and 100 wt %(□)). Ti is the transmittance at 500 nm at pH 9.0, and T is thetransmittance at a selected pH after micelles were stabilized for 24 h.

FIGS. 13A-B show pH-dependent cumulative ADR release from the mixedmicelles comprising polyHis/PEG and PLLA/PEG (PLLA/PEG content in themixed micelles: (FIG. 13A) 0 wt %; pH 8.0 (●); pH 7.4 (▪); pH 7.2 (▴);pH 7.0 (▾); pH 6.8 (♦); pH 6.2 (□); pH 5.0 (Δ). (FIG. 13B) 0 wt % (●);10 wt % (▪); 25 wt % (▴); and 40 wt % (▾) after 24 h.

FIG. 14 shows the cytotoxicity of blank micelles against MCF-7 cells atpH 7.4 after 48 h incubation: PLLA/PEG micelles (●); polyHis/PEGmicelles (▪); and mixed polyHis/PEG and PLLA/PEG micelles with PLLA/PEG25 wt % (A).

FIGS. 15A-E show the cytotoxicity of ADR-loaded mixed micelles withPLLA/PEG content of 0 wt % (FIG. 15A), 10 wt % (FIG. 15B), 25 wt % (FIG.15C), 40 wt % (FIG. 15D), and 100 wt % (FIG. 15E) after 48 h incubationat varying pH: pH 7.4 (●); pH 7.2 (▪); pH 7.0 (▴); pH 6.8 (▾); and pH6.6 (♦). Free ADR toxicity is presented as a reference at pH 7.4 (∘); pH7.2 (□); pH 7.0 (Δ) ; pH 6.8 (∇); and pH 6.6 (⋄).

FIGS. 16A-B show the cytotoxicity of (FIG. 16A) free ADR (●),polyHis/PEG-folate micelles (▪), PLLA/PEG-folate micelles (▴), andpolyHis/PEG-folate and PLLA/PEG-folate mixed micelles (25 wt %PLLA/PEG-folate) (▾) at pH 8.0, and of (FIG. 16B) polyHis/PEG-folate andPLLA/PEG-folate mixed micelles comprising 25 wt % PLLA/PEG-folate(closed symbols) or 40 wt % PLLA/PEG-folate (open symbols) at pH 7.4 (●,∘) and pH 6.8 (▪,□) against MCF-7 cells after 48 h incubation.

FIG. 17 shows the cell killing rate against MCF-7 cells treated withpolyHis/PEG-folate and PLLA/PEG-folate mixed micelles (25 wt % ofPLLA/PEG-folate) (●) and free ADR (▪) at pH 6.8. The ADR content in themicelles was adjusted to be equivalent to the free ADR concentration(500 ng/ml) and MCF-7 cells were treated for a selected incubation time.

FIGS. 18A-C show confocal images of MCF-7 cells treated with (FIG. 18A)polyHis/PEG-folate and PLLA/PEG-folate mixed micelles (25 wt % ofPLLA/PEG-folate), (FIG. 18B) PLLA/PEG-folate micelles, and (FIG. 18C)free ADR.

FIGS. 19A-B show the structures of polyHis/PEG-folate (FIG. 19A) andPPLA/PEG-folate (FIG. 19B).

FIGS. 20A-B show characterization of multidrug resistance inMCF-7/DOX^(R) cells. FIG. 20 A shows flow cytometry studies of sensitiveMCF-7 (thick solid line) and DOX-resistant MCF-7 (MCF-7/DOX^(R)) cells(thin solid line). FIG. 20B shows the cytotoxicity of free DOX againstMCF-7 (closed circles) and MCF-7/DOX^(R) (open circles) cells at pH 8.0(●), pH 7.4 (▪), and pH 6.8 (▴) (n=7).

FIGS. 21A-B show confocal microscopy studies on (FIG. 21A) Dox-sensitiveMCF-7, and (FIG. 21B) DOX-resistant MCF-7 (MCF-7/DOX^(R)) cells treatedwith free DOX at pH 6.8

FIGS. 22A-C show the cytotoxicity of DOX loaded with pH-sensitivemicelles (●), polyHis/PEG-folate micelles (▪), PPLA/PEG-folate micelles(▴), free DOX (▾) against MCF-7/DOX^(R) cells at (FIG. 22A) pH 8.0,(FIG. 22B) pH 7.4, and (FIG. 22C) pH 6.8 after 48 hrs incubation (n=7).

FIGS. 23A-B show confocal microscopy studies on DOX-resistantMCF-7/DOX^(R) cells treated with (FIG. 23A) DOX-loaded pH-sensitivemicelles and (FIG. 23B) DOX-loaded PLLA/PEG-folate micelles at pH 6.8.

FIGS. 24A-B show in vivo tumor volume change (FIG. 24A) and body weightchange (FIG. 24B) of human breast MCF-7 carcinoma xenografts in BALB/cnude mice injected intravenously with 10 mg/kg DOX equivalent dose:mixed micelles of polyHis-b-PEG and PLLA-b-PEG at a 75:25 weight ratio(●), PLLA-b-PEG micelles (▪), free DOX (▴), and saline solution (▾);values are means ± the standard deviation.

FIGS. 25A-B show in vivo tumor volume change (FIG. 25A) and body weightchange (FIG. 25B) of human breast MCF-7/DOX^(R) carcinoma xenografts inBALB/c nude mice injected intravenously with 10 mg/kg DOX equivalentdose: mixed micelles of polyHis-b-PEG-folate and PLLA-b-PEG-folate at a75:25 weight ratio (●), mixed micelles of polyHis-b-PEG and PLLA-b-PEGat a 75:25 weight ratio (▪), PLLA-b-PEG-folate micelles (▴), PLLA-b-PEGmicelles (▾), and free DOX (♦); values are means ± the standarddeviation.

FIGS. 26A-B show in vivo tumor volume change (FIG. 26A) and body weightchange (FIG. 26B) of human lung NCI—H358 carcinoma xenografts in BALB/cnude mice injected intravenously with 10 mg/kg DOX equivalent dose:mixed micelles of polyHis-b-PEG-folate and PLLA-b-PEG-folate at a 75:25weight ratio (●), pLLA-b-PEG-folate micelles (▪), and free DOX (▴);values are means ± the standard deviation.

DETAILED DESCRIPTION

Before the present compositions and methods are disclosed and described,it is to be understood that this invention is not limited to theparticular configurations, process steps, and materials disclosed hereinas such configurations, process steps, and materials may vary somewhat.It is also to be understood that the terminology employed herein is usedfor the purpose of describing particular embodiments only and is notintended to be limiting since the scope of the present invention will belimited only by the appended claims and equivalents thereof.

The publications and other reference materials referred to herein todescribe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference. Thereferences discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to a mixed micelle composition containing “a drug” includes amixture of two or more drugs, reference to “an amino-group protectingreagent” includes reference to one or more of such reagents, andreference to “an initiator” includes reference to a mixture of two ormore of such initiators.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

As used herein, “comprising,” “including,” “containing,” “characterizedby,” and grammatical equivalents thereof are inclusive or open-endedterms that do not exclude additional, unrecited elements or methodsteps. “Comprising” is to be interpreted as including the morerestrictive terms “consisting of” and “consisting essentially of.”

As used herein, “consisting of” and grammatical equivalents thereofexclude any element, step, or ingredient not specified in the claim.

As used herein, “consisting essentially of” and grammatical equivalentsthereof limit the scope of a claim to the specified materials or stepsand those that do not materially affect the basic and novelcharacteristic or characteristics of the claimed invention.

As used herein, “polyHis” means poly(L-histidine); “CBZ” meanscarbobenzoxy; “DNP” means dinitrophenyl; “NCA” means N-carboxyanhydride;“DP” means degree of polymerization; “M/I ratio” meansmonomer-to-initiator ratio; “NHS” means N-hydroxysuccinimide; “DCC”means N,N -dicyclohexylcarbodiimide; and “PEG” means poly(ethyleneglycol). As used herein, “ADR” and “DOX” mean adriamycin or doxorubicin.

As used herein, “polyHis5K-b-PEG2K” means a block copolymer of polyHisand PEG wherein the polyHis portion has a molecular weight of about5,000 and the PEG portion has a molecular weight of about 2,000.

As used herein, “administering” and similar terms mean delivering thecomposition to the individual being treated such that the composition iscapable of being circulated systemically to the parts of the body, e.g.,solid tumors, where the pH-sensitive mixed polymeric micelles encounteracidic conditions, relative to the blood, and release the drug. Thus,the composition is preferably administered to the individual by systemicadministration, typically by subcutaneous, intramuscular, or intravenousadministration, or intraperitoneal administration. Injectables for suchuse can be prepared in conventional forms, either as a liquid solutionor suspension or in a solid form suitable for preparation as a solutionor suspension in a liquid prior to injection, or as an emulsion.Suitable excipients include, for example, water, saline, dextrose,glycerol, ethanol, and the like; and if desired, minor amounts ofauxiliary substances such as wetting or emulsifying agents, buffers, andthe like can be added.

Illustrative anticancer drugs that may be administered according to thepresent invention include acivicin, aclarubicin, acodazole, acronycine,adozelesin, alanosine, aldesleukin, allopurinol sodium, altretamine,aminoglutethimide, amonafide, ampligen, amsacrine, androgens, anguidine,aphidicolin glycinate, asaley, asparaginase, 5-azacitidine,azathioprine, Bacillus calmette-guerin (BCG), Baker's Antifol (soluble),beta-2′-deoxythioguanosine, bisantrene HCL, bleomycin sulfate, busulfan,buthionine sulfoximine, BWA 773U82, BW 502U83.HCl, BW 7U85 mesylate,ceracemide, carbetimer, carboplatin, carmustine, chlorambucil,chloroquinoxaline-sulfonamide, chlorozotocin, chromomycin A3, cisplatin,cladribine, corticosteroids, Corynebacterium parvum, CPT-11, crisnatol,cyclocytidine, cyclophosphamide, cytarabine, cytembena, dabis maleate,dacarbazine, dactinomycin, daunorubicin HCl, deazauridine, dexrazoxane,dianhydrogalactitol, diaziquone, dibromodulcitol, didemnin B,diethyldithiocarbamate, diglycoaldehyde, dihydro-5-azacytidine,doxorubicin, echinomycin, edatrexate, edelfosine, eflornithine,Elliott's solution, elsamitrucin, epirubicin, esorubicin, estramustinephosphate, estrogens, etanidazole, ethiofos, etoposide, fadrazole,fazarabine, fenretinide, filgrastim, finasteride. Flavone acetic acid,floxuridine, fludarabine phosphate, 5-fluorouracil, Fluosol®, flutamide,gallium nitrate, gemcitabine, goserelin acetate, hepsulfam,hexamethylene bisacetamide, homoharringtonine, hydrazine sulfate,4-hydroxyandrostenedione, hydrozyurea, idarubicin HCl, ifosfamide,interferon alfa, interferon beta, interferon gamma, interleukin-1 alphaand beta, interleukin-3, interleukin-4, interleukin-6, 4-ipomeanol,iproplatin, isotretinoin, leucovorin calcium, leuprolide acetate,levamisole, liposomal daunorubicin, liposome encapsulated doxorubicin,lomustine, lonidamine, maytansine, mechlorethamine hydrochloride,melphalan, menogaril, merbarone, 6-mercaptopurine, mesna, methanolextraction residue of Bacillus calmette-guerin, methotrexate,N-methylformamide, mifepristone, mitoguazone, mitomycin-C, mitotane,mitoxantrone hydrochloride, monocyte/macrophage colony-stimulatingfactor, nabilone, nafoxidine, neocarzinostatin, octreotide acetate,ormaplatin, oxaliplatin, paclitaxel, pala, pentostatin, piperazinedione,pipobroman, pirarubicin, piritrexim, piroxantrone hydrochloride,PIXY-321, plicamycin, porfimer sodium, prednimustine, procarbazine,progestins, pyrazofurin, razoxane, sargramostim, semustine,spirogermanium, spiromustine, streptonigrin, streptozocin, sulofenur,suramin sodium, tamoxifen, taxotere, tegafur, teniposide,terephthalamidine, teroxirone, thioguanine, thiotepa, thymidineinjection, tiazofurin, topotecan, toremifene, tretinoin, trifluoperazinehydrochloride, trifluridine, trimetrexate, tumor necrosis factor, uracilmustard, vinblastine sulfate, vincristine sulfate, vindesine,vinorelbine, vinzolidine, Yoshi 864, zorubicin, and mixtures thereof.

According to the present invention, one or more drugs, such asanticancer drugs, are incorporated into pH-sensitive mixed polymericmicelles, and then the drug-loaded pH-sensitive mixed polymeric micellesare administered to a warm-blooded animal, such as a human, such thatthe drug-loaded micelles enter into the systemic circulation of theanimal. When the micelles encounter acidic conditions, such as theacidic microenvironment of solid tumors, the micelles release the drug.The released drug is then available to give its pharmacological effect.

According to the present invention, pH-sensitive mixed polymericmicelles comprise a mixture of poly(L-histidine)-poly(ethylene glycol)block copolymer and an amphiphilic polymer. These pH-sensitive mixedpolymeric micelles are stable in the blood, but release a drug carriedin such micelles upon encountering acidic conditions, such as the acidicmicroenvironments of solid tumors. Either or both of thepoly(L-histidine)-poly(ethylene glycol) block copolymer and theamphiphilic polymer can be covalently coupled to a targeting moietyaccording to the present invention. The targeting moiety permits bindingto receptors on the surfaces of cells. In one illustrative embodiment,folate is such a targeting moiety that has been covalently bonded topoly(L-histidine)-poly(ethylene glycol) block copolymer and toamphiphilic polymers. Other targeting moieties known in the art are alsowithin the scope of the present invention. Amphiphilic polymersaccording to the present invention include poly(L-lacticacid)-poly(ethylene glycol) block copolymer, poly(DL-lactic-co-glycolicacid), and ABA block copolymers, such as poly(ethyleneoxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymer, andmixtures of any of the above.

Synthesis of Poly(L-histidine) and Block Copolymers

The imidazole ring has an electron lone pair on the unsaturated nitrogenthat endows polyHis with an amphoteric nature byprotonation-deprotonation. Its pK_(b) value and pH-solubility dependenceare influenced by the molecular weight (MW). D. W. Pack et al., Designof imidazole-containing endosomolytic biopolymers for gene delivery, 67Biotech. Bioeng. 217-223 (2000); R. C. Bohinski, Modern Concepts inBiochemistry 4-24, 61 (4^(th) ed. Allyn and Bacon: Boston, 1983). Thepolymer with a MW higher than 10,000 g/mole is water-soluble below pH6.0 as a result of protonation. D. W. Pack et al., supra. In theprotonated state, polyHis forms particles (120-200 nm) with anioniclipids, which demonstrated metal chelating properties. S. General & A.F. Thunemann, pH-sensitive nanoparticles of poly(amino acid) dodecanoatecomplexes, 230 Int'l J. Pharm. 11-24 (2001); M. P. McCurdie & L. A.Belfiore, Solid-state complexes of poly(L-histidine) with metalchlorides from the first row of the d-block, 37 J. Polym. Science: PartB: Polymer Physics. 301-310 (1999).

Recently, the probable fusogenic activity of polyHis, which disrupts theenvelope membrane of acidic subcellular compartments such as endosomesand lysosomes, has drawn much attention for the design of non-viral genedelivery carriers. Plasmid DNA (pDNA) was incorporated into cationicpolymeric carriers grafted with short polyHis side chains, P. Midoux &M. Monsigny, Efficient gene transfer by histidylated polylysine/pDNAcomplexes, 10 Bioconjugate Chem. 406-411 (1999); D. Putnam et al.,Polymer-based gene delivery with low cytotoxicity by a unique balance ofside-chain termini, 98 Proc. Nat'l Acad. Sci. USA 1200-1205 (2001); J.M. Benns, J. S. Choi, R. I. Mahato, J. S. Park, S. W. Kim, pH-sensitivecationic polymer gene delivery vehicle:N-Ac-poly(L-histidine)polyHis-graft-poly(L-lysine) comb shaped polymer,11 Bioconjugate Chem. 637-645 (2000), and the resulting complexdemonstrated an increase in gene transfection efficacy. This result wasexplained by assuming that the endosomal membrane had been disrupted bythe proton-sponge effect of imidazole groups, facilitating the releaseof the DNA/polymer complex into the cytoplasm. In addition, polyHis hasbeen shown to fuse with lipid bilayers upon protonation of imidazolegroups as a result of interaction with negatively charged membranephospholipids. C. Y. Wang & L. Huang, Polyhistidine mediates an aciddependent fusion of negatively charged liposomes, 23 Biochemistry4409-4416 (1984). However, the use of polyHis as a gene carrier has beenlimited because polyHis does not form complexes with pDNA at neutral pHlevels. Instead, poly(L-lysine) has been partially substituted withhistidyl (or imidazole) residues. These histidylated poly(L-lysine)/pDNAcomplexes remarkably improve the transfection efficiency in vitro. P.Midoux & M. Monsigny, supra; D. Putnam et al., supra; J. M. Benns etal., supra.

Despite the potential of polyHis, its synthesis has restricted itsapplication. The first synthetic method, ring-opening polymerization ofN^(im)-benzyl-L-histidine N-carboxy-anhydride, was reported by A.Patchornik et al., poly-L-histidine, 79 J. Am. Chem. Soc. 5227-5230(1957). However, difficulties in protecting the imidazole group by theformation of a stable L-histidine N-carboxyanhydride, and purifyingN-carboxyanhydride, were serious problems. J. N. Cha et al., Biomimeticsynthesis of ordered of silica structures mediated by blockcopolypeptides, 403 Nature 289-292 (2000); D. Poland & H. A. Scheraga,Theory of noncovalent structure in polyamino acids, in Poly-α-aminoAcids 391-398 (Marcel Dekker, Inc., New York 1967). Since then, fewpapers on the synthesis of polyHis have been published, and methods forcontrolling the molecular weight (MW) of polyHis have not beenavailable. However, a relatively higher molecular weight polyHis (up to20,000 g/mole) has been commercialized. Recently, a few investigatorshave attempted to synthesize polyHis by peptide-synthesis methods, suchas solid-phase (p-alkoxybenzyl alcohol resins) or liquid-phase(dimethylformamide) peptide synthesis, but the MW was relatively low,about 2,800 g/mole. D. Putnam et al., supra; J. S. Choi et al., Novelmacromolecular self-organization of poly(ethyleneglycol)-block-poly(L-histidine): pH-induced formation of core-shellnanoparticles in aqueous media, 22 Bull. Korean Chem. Soc. 261-262(2001).

EXAMPLES 1-9

Examples 1-9 describe a method of making poly(L-histidine) (“polyHis”)by ring-opening polymerization of L-histidine N-carboxyanhydride, theimidazole amine group of which was protected by a dinitrophenyl group.The synthesis of N^(im)-DNP-L-histidine NCA from L-histidine issummarized in FIG. 1, and the synthesis of polyHis fromN^(im)-DNP-L-histidine NCA is summarized in FIG. 2. The resultingpolyHis polymer (MW: 5,000 g/mole) was coupled to poly(ethylene glycol)(MW: 2,000 g/mole) via an amide linkage using the N,N′-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS)-mediated reaction.Poly(L-histidine)-poly(ethylene glycol) diblock copolymers(polyHis-b-PEG) were prepared and used for the construction of polymericmicelles responding to local pH changes in the body. The block copolymerin dimethylsulfoxide (DMSO) formed polymeric micelles on diafiltrationagainst a borate buffer at pH 8. Dynamic light scattering (DLS) andatomic force microscopy (AFM) showed the micelles were spherical, havingdiameters of about 114 nm, with a unimodal distribution. The criticalmicelle concentration (CMC) at pH 8.0 was 2.3 mg/L. The CMC increasedmarkedly on decreasing the pH of the diafiltration medium below pH 7.2.Micelles prepared at pH 8.0 were gradually destabilized below pH 7.4, asevidenced by a slight increase in light transmittance, an alteration insize distribution, and a decrease in the pyrene fluorescence intensity.Therefore, the ionization of the polyHis block forming the micelle coredetermined the pH-dependent CMC and stability.

EXAMPLE 1 Synthesis of N^(α)-CBZ-L-histidine

L-Histidine monohydrochloride monohydrate (Sigma Chemical Co., Inc., St.Louis, Mo.) was purified by recrystallization from water/ethanol (7/3v/v) at 0° C. The purified L-histidine monohydrochloride monohydrate (15g) in ammonia water (28 wt %, 100 mL) was cooled to 0° C. Benzylchloroformate (15 mL; Aldrich Chemical Co., Inc., Milwaukee, Wis.) wasadded dropwise to the solution while stirring continuously, whichresulted in a white suspension. Stirring was continued for 1 h, followedby addition of an excess amount of diethyl ether (J. T. Baker, Deventer,Netherlands) to the reaction mixture and mixing for more 3 h. Theimpurities (excess benzyl chloroformate and by-products) were extractedtwice with diethyl ether. Ammonia was removed from the aqueous phase,and the volume was increased to 100 ml with deionized water. Afterfiltering, the filtrate was acidified by adding dilute sulfuric acidslowly with stirring until precipitation began. The isolated precipitate(mainly N^(α)-CBZ-L-histidine), collected by filtration, was dissolvedin acetic acid (50 mL) to remove any insoluble residuals. The productNO-CBZ-L-histidine was recrystallized from excess ethanol at 0° C.,yield 62%. The product of this and other examples were analyzed using ¹HNMR (300 MHz, 6 wt % sample solution) and by FT-IR (Perkin Elmer IR2000, KBr pellet). ¹H-NMR (D₂O, TMS): δ 8.09 (—N═CH—), δ 7.43 (protonson benzyl of CBZ), δ 6.05 ((—N—CH═C—), δ 5.41 (—COOCH₂—), d 4.52 (—CH—)and d 3.10 (—CH₂—).

EXAMPLE 2 Synthesis of N^(α)-CBZ-N^(im)-DNP-L-histidine

A solution of Na-CBZ-L-histidine (7 g), prepared according to theprocedure of Example 1, in distilled water (100 mL) containing sodiumbicarbonate (8.4 g, to delay the reaction of 2,4-dinitrofluorobenzenewith water; Sigma Chemical) and 0.5 M sodium hydroxide (NaOH, anactivation agent) was cooled to 0° C. and 2,4-dinitrofluorobenzene (5mL; Aldrich Chemical) in 1,4-dioxane (50 mL) was added. The reactionmixture was vigorously stirred at 0° C. for 8 h, then acidified with 3 NHCl aqueous solution to precipitate the product. The precipitate wasfiltered, washed with a small quantity of water and then with ethanol.After dissolving NW-CBZ-N^(im)-DNP-L-histidine in tetrahydrofuran (THF;Sigma Chemical), a 2-fold excess of petroleum ether (J. T. Baker) wasadded with stirring to reprecipitate Na-CBZ-N^(im)-DNP-L-histidine. Thesuspension was left undisturbed at 0° C. for one day, followed byfiltration. This process was repeated. The final product was dried invacuo for two days; yield 56%. ¹H-NMR (DMSO-d₆ with TMS): δ 7.86-8.99(protons on phenyl group, DNP), δ 8.05 (—N═CH—), δ 7.43 (protons onbenzyl group, CBZ), δ 6.94 (—N—CH═C), d 5.41 (—COOCH₂—), d 4.52 (—CH—)and d 3.10 (—CH₂—).

EXAMPLE 3 Synthesis of N^(im)-DNP-L-histidine carboxyanhydridehydrochloride

N^(α)-CBZ-N^(im)-DNP-L-histidine (5 g), prepared according to theprocedure of Example 2, was dried over phosphorus pentoxide in vacuo,dissolved in anhydrous THF (35 mL) and thionyl chloride (0.8 mL; Fluka,Buchs, Switzerland) was added. The mixture turned opaque and highlyviscous. The reaction was allowed to occur at room temperature,resulting in a clear solution in a few minutes. After another 40 min, anexcess of anhydrous diethyl ether (8-fold) was added to precipitate theproduct and the precipitate was filtered and dried in vacuo. To removeimpurities, the product was dissolved in nitromethane (Aldrich Chemical)at room temperature and insoluble impurities removed by filtration.After stirring the solution in the presence of the activated carbon(dried in vacuo at 200° C. for three days), the solution was filteredthree times to remove the activated carbon. N^(im)-DNP-L-histidinecarboxyanhydride hydrochloride was crystallized by adding an 8-foldexcess amount of anhydrous ethyl ether to the nitromethane solution.This process was repeated to obtain pure the pure N-carboxyanhydride(NCA). The crystals were filtered and dried for two days in vacuo in thepresence of phosphorus pentoxide and NaOH powder; yield 67%. So-purifiedL-histidine-NCA was used for polymerization within 3 days. ¹H-NMR(DMSO-d₆with TMS): δ 7.86-8.99 (protons on phenyl group, DNP), δ 8.05(—N═CH—), δ 7.10 (s; —NH), d 6.94 (—N—CH═C—), d 4.52 (—CH—), and d 3.10(—CH₂—), respectively ; no CBZ group peak. FT-IR spectrum: 1780-1790cm⁻¹ (N—C═O), 3340-3350 cm⁻¹ (NH)

EXAMPLE 4 Polymerization of N^(im)-DNP-L-histidine carboxyanhydridehydrochloride

N^(im)-DNP-L-histidine NCA hydrochloride (1.4 g), prepared according tothe procedure of Example 3, was dissolved in purified anhydrousdimethylformamide (DMF; J. T. Baker) (30 mL) and a predetermined amountof initiator (hexylamine or isopropylamine (Sigma Chemical) purifiedbefore use) was added to obtain different molecular weights of polymers.The polymerization proceeded at room temperature with evolution ofcarbon dioxide for 72 h (FIG. 2). An excess of 0.1 N HCl (600 mL) wasadded to precipitate poly(N^(im)-DNP-L-histidine). After filtration,poly(N^(im)-DNP-L-histidine) was dried in vacuo over phosphoruspentoxide for two days; yield 90%. The degree of polymerization (DP) ofthe polymers was estimated from the ¹H-NMR spectrum (DMSO-d₆ with TMS)using the integration ratio of the peaks from the repeating unit (—CH—)and the initiator (—CCH₃). ¹H-NMR spectrum: δ 7.86-8.99 (protons onphenyl group, DNP), δ 7.91 (—N═CH—), δ 6.94 (N—CH═C—), d 4.85 (—CH—), d3.85 (—CH—NH₂), d 3.10-3.26 (—CH₂—) and d 1.06 (—CCH₃—).

N^(α)-CBZ-N^(im)-DNP-L-histidine NCA.HCl was not soluble in most organicsolvents used for the polymerization of amino acid NCAs, with theexception of DMF. The ring opening polymerization ofNu-CBZ-N^(im)-DNP-L-histidine NCA.HCl in DMF was performed usingdifferent molar ratios of the monomer to initiator (isopropylamine orn-hexylamine) (M/I ratio). Table 1 shows that the degree ofpolymerization (DP) of poly(N^(im)-DNP-L-histidine) depended on the M/Iratio and the type of initiator. The DP of the polymer increased withincreasing M/I ratio except for the high M/I ratios of 40:1 and 50:1.The primary amine as an initiator has higher reactivity with NCA thansecondary or tertiary amine and induces faster initiation ofpolymerization. However, in this example high M/I ratios of 40:1 and50:1 produced rather low molecular weight polymers. This observation isnot well understood, but could be related to the diluted initiatorconcentration, which resulted in a longer time for polymerization, andto residual HCL salt in NCA. A molecule of L-histidine NCA has twonitrogens in the imidazole ring both of which are HCL salt-attachable,and the initiation may be influenced by the residual salts. A.Patchornik et al., supra; H. R. Kricheldorf,α-Aminoacid-N-carboxy-anhydrides and related heterocycles 93-95, 2(Springer-Verlag Berlin Heidelberg New York 1987); W. N. E.Dijk-Wolthuis et al., Synthesis and characterization of poly-L-lysinewith controlled low molecular weight, 198 Macromol. Chem. Phys.3893-3906 (1997). TABLE 1 Degree of polymerization of polyHis with M/Imolar ratio and initiator (n = 5). Primary M/I(N-carboxyanhydrides/Initiator) molar ratio Initiator 20/1 30/1 35/140/1 50/1 n-Hexyl- 16.2 ± 3.4 16.2 ± 3.4 28.3 ± 4.4 28.5 ± 3.5 12.3 ±2.6 amine Isopropyl- 20.3 ± 3.3 32.4 ± 3.5 33.5 ± 3.6 24.5 ± 3.1 12.6 ±2.9 amine

For the M/I ratios of 20:1-35:1, almost all initiator molecules areexpected to be incorporated into the growing chains. For n-hexylamine,the experimental DP was lower than the theoretical DP calculated fromthe M/I ratio. However, the DP of poly(N^(im)-DNP-L-histidine) initiatedby isopropylamine was close to the theoretical DP and slightly higherthan theoretical expectation. This result is probably due to therelatively slower initiation and faster propagation rates byisopropylamine compared to n-hexylamine. H. R. Kricheldorf, supra. Aslower initiation rate has often been observed in the case of secondaryor tertiary amines that result in higher MW. A. Patchornik, supra; H. R.Kricheldorf, supra; W. N. E. Dijk-Wolthuis et al., supra. As shown inTable 1, the highest DP (33.5±3.6) of the polymer was obtained when themolar ratio of NCA to isopropylamine was 35:1.

EXAMPLE 5 Synthesis of poly(N^(im)-DNP-L-histidine) and PEG blockcopolymer

Monocarboxylic acid-PEG was prepared as described in S. Zalipsky & G.Barany, Facile synthesis of α-hydroxy-ω-carboxymethylpolyethylene oxide,5 J. Bioactive and Compatible Polymers 227-231 (1990). The preactivationof PEG (NHS-PEG) was performed with PEG (1 mole), NHS (1.5 moles), andDCC (1.25 moles) in methylene chloride (J. T. Baker) at roomtemperature. The coupling reaction between poly(N^(im)-DNP-L-histidine)and NHS-PEG (1:1 functional group ratio) was carried out in THF for twodays at room temperature. After reaction, the block copolymer wasprecipitated in a mixed solvent (THF/n-hexane) and filtered. Thecoupling of poly(N^(im)-DNP-L-histidine) and PEG was confirmed by thetransfer of peak at δ 3.85 (—CH—NH₂) to δ 4.85 (—CH—NH—CO—). The polymerwas dried in vacuo for 2 days.

EXAMPLE 6 Deblocking of DNP Group

2-Mercaptoethanol (25 mL; Sigma Chemical) was added to thepoly(N^(im)-DNP-L-histidine)-PEG diblock copolymer (2 g), preparedaccording to the procedure of Example 5, in DMF (80 mL) (FIG. 2).Deprotection was complete within one day at room temperature. Thesolution was added to diethyl ether (800 mL) at 0° C. to precipitatepolymer, while excess 2-mercaptoethanol and DNP-mercaptoethanol remainedsoluble. The polymer was filtered, washed with diethyl ether, and driedin vacuo for two days. For further purification, the polymer wasdissolved in a minimum volume of 3 N HCl and stored in a 0° C. for oneday. After precipitation of DNP was complete, the solution was filtered(0.45 μm) and the product was precipitated after adding excess acetoneto the solution, and then dried in vacuo for 2 days. Dialysis(Spectra/Por; MWCO 5,000) was used to remove uncoupled polymers. Beforesubsequent experiments, the HCl salt of polyHis (1 mmole) in DMSO wasconverted to the free base by treatment with triethylamine (TEA; SigmaChemical) (2 mmoles) for 3 h at room temperature; the free base wasrecrystallized from ethanol and dimethylsulfoxide (DMSO; J. T. Baker)(10:1).

The removal of DNP was confirmed by ¹H-NMR (DMSO-d₆ with TMS): 8 7.90(—N═CH—), δ 6.92 (N—CH═C), 8 4.85 (-CH—), δ 5.03 (OCH₂CONH,poly(ethylene glycol), δ 3.62-3.81 (protons on repeating units,poly(ethylene glycol), δ 3.10-3.26 (—CH₂—) and δ 1.06 (—CCH₃—). Thediblock copolymers used for further micelle study were polyHis (MW:5,000 g/mole)-block-poly(ethylene glycol) (MW: 2,000 g/mole) (denoted aspolyHis5K-b-PEG2K) and polyHis (MW: 3,100 g/mole)-block-poly(ethyleneglycol) (MW: 2,000 g/mole) (polyHis3K-b-PEG2K). The conjugation yieldswere 92 wt % and 86 wt %, respectively.

EXAMPLE 7 Titration of PolyHis-b-PEG Block Copolymers

The polymers and NaCl (control) were dissolved in 35 mL of deionizedwater (30 μmole/L) and the solution was adjusted to pH 12 with 1 M NaOH.The diluted solution was titrated by stepwise addition of 1 N HCLsolution to obtain the titration profile. D. W. Urry et al., Comparisonof electrostatic- and hydrophobic-induced pKa shifts inpolypentapeptides. The lysine residue, 225 Chem. Phys. Lett. 97-103(1994). The average value from triplicate titrations was plotted.

It has been documented in literature that histidine residues in proteinssignificantly contribute to the protein buffering capacity atphysiological pH level. A. Patchornik et al., supra. The acid-basetitration profiles of the polyHis homopolymer and polyHis-b-PEG blockcopolymers are presented in FIG. 3. All of the polymer solutionsexhibited a buffering pH region of pH 4-9. The titration curve confirmedthat the polymers with a higher molecular weight polyHis block had ahigher buffering capacity (FIG. 3A) in the physiological pH range of pH5.5-8.0 due to the higher concentrations of imidazole rings.PolyHis5K-b-PEG2K and polyHis3K-b-PEG2K had inflexion points around pH7.0 (pK_(b)). PolyHis5K and PolyHis3K showed pKb values around pH 6.5.The pK_(b) shift of the block copolymer compared to the polyHishomopolymer may be due to increased hydration of the PEG. D. W. Urry etal., Comparison of electrostatic- and hydrophobic-induced pKa shifts inpolypentapeptides. The lysine residue, 225 Chem. Phys. Lett. 97-103(1994).

EXAMPLE 8 PolyHis-b-PEG Copolymer Micelles

Each polymer (20 mg) prepared according to the procedures of Examples1-7 dissolved in DMSO (20 mL) was transferred to a pre-swollen dialysismembrane tube (Spectra/Por; MWCO 15,000) and dialyzed against HCl (orNaOH)—Na₂B₄O₇ buffer solution (pH 5.0-8.0, ionic strength=0.1) for 24 h.The outer phase was replaced with fresh buffer solution at 1, 2, 4, 6,and 10 h. The solution was subsequently lyophilized after filteringthrough a 0.8 μm syringe filter. The yield (wt %) of micelles wascalculated by weighing the freeze-dried micelle powder.

The deprotonated polyHis at pH 8.0 is hydrophobic, while PEG is solublein water at all pH's. This amphiphilicity at pH 8.0 was responsible forthe formation of polymeric micelles. Lowering the solution pH below thepK_(b) can affect the micellar structure because protonation convertsthe hydrophobic polyHis to a more hydrophilic block.

The polyHis-b-PEG block copolymer micelles were prepared bydiafiltration of polymer solution in DMSO against a pH 8.0 medium. Theyields (wt %) of micelle formation were 90-93 wt % for the blockcopolymer with polyHis5K and 28 wt % for polyHis3K-b-PEG2K.

To evaluate the micellar stability, the time dependent turbidity changeof each micelle was measured at pH 8.0 using a Varian CARY 1E UV/VISspectrophotometer (FIG. 4). PolyHis3K-b-PEG2K micelles exhibitedinstability over time at pH 8.0 as shown by increasing lighttransmittance, while the stability of polyHis5K-b-PEG2K micelles at pH8.0 was maintained for two days. The instability of polyHis3K-b-PEG2K atpH 8.0 was probably due to short polyHis block length and indicates thatthere may be a polyHis length somewhere between MW 3000 and MW 5000 atwhich stability increases.

Micelle formation was monitored by fluorometry in the presence of pyreneas a fluorescent probe. A stock solution of pyrene (6.0×10⁻² M; SigmaChemical) was prepared in acetone and stored at 5° C. until further use.For the measurement of steady-state fluorescence spectra, the pyrenesolution in acetone was added to de-ionized water to give a pyreneconcentration of 12.0×10⁻⁷ M. The solution was then distilled undervacuum at 60° C. for 1 h to remove acetone from the solution. Theacetone-free pyrene solution was mixed with the solution of polymericmicelles, the concentration of which ranged from 1×10⁻⁴ to 1.0 g/L. Forpreparation of polymeric micelle solutions, a freeze-dried micellesample was dispersed in HCl (or NaOH) —Na₂B₄O₇ buffer solution (pH5.0-8.0, ionic strength=0.1). The initial pH of each micelle solutionused for fluorescence study was tuned to the diafiltration pH used formicelle fabrication.

The final concentration of pyrene in each sample solution was 6.0×10⁻⁷ M(its solubility limit in water at 22° C.). The pyrene emission at 339 nmwas recorded. The CMC was estimated by plotting I₁ (intensity of firstpeak) of the emission spectra profile against of the log of the micelleconcentration.

Pyrene strongly fluoresces in a non-polar environment, while in a polarenvironment it shows weak fluorescence intensity. The change of totalemission intensity vs. polymer concentration indicates the formation ofmicelle or the change from micelle to unimer (dissociated polymer fromdisrupted micelle). C. M. Marques, Bunchy micelles, 13 Langmuir1430-1433 (1997). FIG. 5A shows the change in intensity (I₁) of thefirst peak in the emission spectra plotted against polymerconcentration. The CMC value was determined from the crossover point atlow concentrations. The CMC of polyHis5K-b-PEG2K at pH 8.0 was 2.3 μg/mL(FIG. 5B) while that of polyHis3K-b-PEG2K was 62 μg/mL. This resultsupports the general propensity for a more hydrophobic block or a longerhydrophobic block to reduce the CMC.

Since polyHis block is a polybase, the effect of the diafiltration pH onthe CMC was examined and the results are presented in FIG. 6. At adiafiltration pH between 8.0 and 7.4, the CMC of polyHis5K-b-PEG2Kmicelle increased slightly with decreasing pH. However, the CMC wassignificantly elevated below pH 7.2. It is evident that the protonationof the imidazole group in the copolymer at lower pH level causes areduction in hydrophobicity, leading to an increase in CMC. In addition,below pH 5.0 (typically pH 4.8), the CMC of polyHis5K-b-PEG2K micellecould not be detected. Taken together, at pH 8.0 the less protonatedpolyHis constitutes the hydrophobic core in the micellar structure, butin the range of pH 5.0-7.4 the polymer produced less stable micelles.This pH-dependent stability due to the protonation of the hydrophobiccore is consistent with V. Biitiin et al., Unusual aggregation behaviorof a novel tertiary amine methacrylate-based diblock copolymer:formation of micelles and reverse micelles in aqueous solution, 120 J.Am. Chem. Soc. 11818-11819 (1998). In the latter case, the“schizophrenic” AB block copolymers were composed of2-(diethylamino)ethyl methacrylate (DEA) and 2-(N-morpholino)ethylmethacrylate (MEMA). The micelles from these block copolymers showedflip-flop self-assembly at pH 6.0-8.0, depending on the pK_(a) value ofeach block. DEA-core micelles formed at pH 8.0 and disintegrated tounimers below pH 7.0, while MEMA-core micelles formed at pH 6.7 andreverted to unimers below pH 6.0.

The size of the micelles formed from polyHis5K-b-PEG2K was measured byphoton correlation spectroscopy (PCS) using a Zetasizer 3000 (MalvernInstruments) with a He—Ne laser beam at a wavelength of 633 nm at 25° C.and a fixed scattering angle of 90°. The polymeric micelles (0.1 g/L)were exposed to different pH's for 24 h before measurement of theparticle size and particle distribution. The particle size was 114 nmbased on the intensity-average diameter with a unimodal distribution(FIG. 7A), and the ratio of weight average particle size to numberaverage particle size was 1.2. The relatively large size ofpolyHis5K-b-PEG2K micelles may be the result of the diafiltrationconditions. Kohori et al., Process design for efficient and controlleddrug incorporation into polymeric micelle carrier system, 78 J. Control.Rel. 155-163 (2002), reported that the size of polymeric micellescomposed ofpoly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(DL-lactide)changed from 50 nm to 250 nm under different diafiltration conditions.The large size of polymeric micelles (over 100 nm) is most probably dueto the formation of secondary aggregates that are clusters of individualsingle micelles. S. B. La et al., Preparation and characterization ofthe micelle-forming polymeric drug indomethacin-incorporatedpoly(ethylene oxide)-poly(beta-benzyl-L-aspartate) block copolymermicelle, 85 J. Pharm. Sci. 85-90 (1996). There is a probability thatPolyHis5K-b-PEG2K micelles exist in a solution as secondary micelles.However, the polyHis5K-b-PEG2K micellar shape was regular and sphericalas visualized by atomic force microscopy (AFM) (FIG. 7B). The AFM samplewas prepared by casting a dilute micelle solution (10 mg/L, NaOH—Na₂B₄O₇buffer solution, pH 8.0, ionic strength=0.1) on a glass slide, which wasthen dried in vacuo and observed by AFM (ThermoMicroscopes Explorer withECU-plus electronics, Santa Clara, Calif.). The morphology of polymericmicelle was further evident from DSL measurements. DLS measurements wereperformed with an argon ion laser system adjusted to a wavelength of 488nm. Each sample (0.1 g/L) was filtered through a 0.45-μm filter directlyinto a pre-cleaned 10 mm diameter cylindrical cell. The scattering anglewas varied from 300 to 1500 and the temperature was controlled at 25° C.The particle diffusivity (or size) was calculated using theStokes-Einstein equation, defined as R_(H)=(k_(B)×T)/(6×π×η×D₀), whereR_(H)=hydrodynamic radius, k_(B)=Boltzmann constant, T=absolutetemperature, η=solvent viscosity, D₀=diffusion coefficient at infinitedilution. The angular dependent diffusivity (or size) was obtained toestimate the micelle shape. FIG. 8 shows that the relaxation rates (r)were proportional to the square of the scattering vector (K=4pn₀sin(q/2)/1₀), where there was n₀=solvent refractive index,1₀=wavelength, and q=scattering angle. Considering that thetranslational diffusion coefficient (D) of spherical particles isindependent of detection angles, R. Xu et al., Light-scattering study ofthe association behavior of styrene-ethylene oxide block copolymers inaqueous solution, 24 Macromolecules 87-93 (1991), the D-values ofparticles can be calculated from the equation r═DK². R. Xu et al.,supra; M. Iijima et al., Core-polymerized reactive micelles fromheterotelechelic amphiphilic block copolymers, 32 Macromolecules1140-1146 (1999). As shown in FIG. 8, D has no angular dependency andthis confirms spherical micelles.

EXAMPLE 9 pH-Sensitivity of Micelles

The light transmittance of solutions was measured using a Varian CARY 1EUV/VIS spectrophotometer. pH was measured with a Fisher Accument Model15 pH Meter. The pH-sensitivity was determined by measuring thepH-dependent light transmittance of the micellar solution, using aconcentration of 0.1 g/L, initially at pH 8.0 (NaOH—Na₂B₄O₇ buffersolution, ionic strength=0.1). The pH was gradually decreased by adding0.01 N HCl solution.

To verify the initial stability of micelles formulated at pH 8.0, theturbidity change of micelle solutions (0.1 g/L) at pH 8.0 (preparationpH) was determined from the light transmittance at λ=500 nm. ThepH-dependent stability of micelles was measured by the transmittancechange after the micelle samples were equilibrated with different pHbuffer solutions (HCL or NaOH—Na₂B₄O₇ buffer, pH 6-8) for 24 h. Thechange in the pyrene fluorescence intensity of first peak (I₁) wasmeasured after each sample was exposed to a different pH for 24 h.

A noticeable increase in light scattering intensity was observed afterdialyzing the block copolymer solutions at pH 8.0, while the unimersolution in DMSO was practically transparent. This indicates theformation of polymeric micelles after diafiltration. The micellestability was confirmed by the measurement of transmittance of themicelle solution. FIG. 9 shows the transmittance change of the polymericmicelle solution as a function of pH. As the pH of the micelle solutiondecreased from the diafiltration conditions (pH 8, ionic strength 1.0),the transmittance increased. In particular, this increase was prominentfrom pH 7.4 to 6.0, reaching a plateau around pH 6.4. This resultimplies that polyHis5K-b-PEG2K micelles start dissociation from pH 7.4.

FIG. 10 shows the changes in the particle size distribution of themicelles as a function of pH. The instability of the micelles onreduction of pH resulted in release of polyHis5K-b-PEG2K unimers thatcreated a bimodal size distribution with existing micelles. Theintensity of the peak at size 2-30 nm gradually increased with micelledisruption at a lower pH. Below pH 7.4, an abrupt increase in theintensity of small sized particles was apparent, which is consistentwith the transmittance change of the micelle solution at this pH (FIG.9).

The micropolarity of pyrene as a function of pH was monitored to obtaina more detailed picture of the interior structural change of polymericmicelles. On reducing the solution pH, pyrene molecules in the polymericmicelles underwent a sharp increase in polarity, which was reflected bya decline in the fluorescence intensity of pyrene. FIG. 11 shows thatthe polyHis5K-b-PEG2K micelles prepared at pH 8.0 exhibit a sudden dropin fluorescence intensity at pH 7.0-7.4, followed by a gradual fall to apH level of 6.0. It is apparent that this reduction of the fluorescenceintensity was due to the instability of hydrophobic core and the causeof the dissociation of micelles is attributed to the protonation of theimidazole ring.

These pH-sensitive micelles will have wide utility in pharmaceuticalapplications, such as solid tumor treatment. For instance, the acidityof solid tumors is distinguishable from normal tissues. G. Kong et al.,Hyperthermia enables tumor-specific nanoparticle delivery: effect ofparticle size, 60 Cancer Res. 4440-4445 (2000). The production ofproduction of lactic acid under hypoxic conditions and the hydrolysis ofATP in an energy deficient environment are partially responsible for theacidic microenvironment of solid tumors. The blood pH is 7.4, while theextracellular pH (pH_(e)) of most solid tumors in patients measured byinvasive microelectrodes ranged from pH 5.7 to pH 7.8, with a mean valueof pH 7.0, wherein 80% of measured values are below pH 7.2. M. Stubbs etal., Causes and consequences of tumour acidity and implications fortreatment, 6 Opinion 15-19 (2000).

Therefore, a polyHis5K-b-PEG2K block copolymer was synthesized and usedto prepare pH-sensitive micelles. The critical micelle concentration was2.3 mg/L at pH 8.0. The stability of the micelles depended on thehydrophobicity at a specified pH. The hydrophobic imidazole group in thehistidine repeat unit becomes hydrophilic as a result of protonation ofthe amine group at lower pH. This pH sensitivity makes the micelles moreeffective for treating solid tumors by providing a switching mechanismfor the release of drugs. Conventional pH-sensitive liposomes cannotdistinguish differences in pH that are less than 1 pH unit. O. V.Gerasimov et al., Cytosolic drug delivery using pH- and light-sensitiveliposomes, 38 Adv. Drug Deliv. Rev. 317-338 (1999). PolyHis5K-b-PEG2Kmicelles can provide the triggered release of an anticancer drug attumor extracellular pH (pH_(e), ≦pH 7.2) levels by physicaldestabilization of long-circulating polymeric micelles, G. Molineaux,Pegylation: engineering improved pharmaceuticals for enhanced therapy,Cancer Treat Rev. Suppl. A 13-16 (2002), to give higher localconcentrations of the drug at tumor sites (targeted high-dose cancertherapy).

Mixed Polymeric Micelles

Polymeric micelles based on poly(L-histidine) (polyHis) as apH-sensitive polybase, were further investigated as a pH-sensitiveanticancer drug carrier. Here, polyHis was selected due to themultifunctionality to pH sensitivity, A. Patchornik et al., supra,biodegradability, and fusogenic activity, J. M. Benns et al., supra; D.Putnam et al., supra; C. Y. Wang & L. Huang, supra. The micelles werecomposed of polyHis/PEG and poly(L-lactic acid) (PLLA)/PEG blockcopolymer with or without folate conjugation, the folate conjugationaimed at folate-receptor-mediated endocytosis. S. D. Weitmann et al.,Distribution of the folate receptor GP38 in normal and malignant celllines and tissues, 52 Cancer Res. 3396-3401 (1992); J. F. Ross et al.,Differential regulation of folate receptor isoforms in normal andmalignant tissues in vivo and in established cell lines. Physiologic andclinical implications, 73 Cancer 2432-2443 (1994); P. S. Low & R. J.Lee, Folate-mediated tumor cell targeting of liposome-entrappeddoxorubicin in vitro, 1233 Biochem. Biophys. Acta 134-144 (1995); J. A.Reddy & P. S. Low, Folate-mediated targeting of therapeutic and imagingagents to cancers, 15 Crit. Rev. Ther. Drug Carrier Syst. 587-627(1998). The synthetic chemistry and physicochemical property ofpolyHis/PEG micelles were described in Examples 1-9.

EXAMPLES 10-15

Novel pH-sensitive polymeric mixed micelles composed ofpoly(L-histidine) (polyHis; MW 5,000)/PEG (MW 2,000) and poly(L-lacticacid) (PLLA) (MW 3,000)/PEG (MW 2,000) block copolymers with or withoutfolate conjugation were prepared by diafiltration. The micelles wereinvestigated for pH-dependent drug release, folate receptor mediatedinternalization and cytotoxicity with MCF-7 cells in vitro. ThepolyHis/PEG micelles showed accelerated adriamycin release as decreasingpH from 8.0. When the cumulative release for 24 hrs was plotted as afunction of pH, the gradual transition in release rate appeared in a pHrange from 8.0 to 6.8. To target triggering pH of polymeric micelles tomore acidic tumor extracellular pH while improving the micelle stabilityat pH 7.4, PLLA/PEG block copolymer was blended with polyHis/PEG to formmixed micelles. This blending shifted the triggering pH to a lowervalue. Depending on the amount of PLLA/PEG, the mixed micelles weredestabilized in the pH range of 7.2-6.6 (triggering pH for adriamycinrelease). When the mixed micelles were conjugated with folic acid, thein vitro results demonstrated that the micelles were more effective intumor cell kill due to accelerated drug release and folatereceptor-mediated tumor uptake. In addition, polyHis afterinternalization was proven to be effective for cytosolic ADR delivery byits fusogenic activity. This approach is expected to treat solid tumorsin vivo.

EXAMPLE 10 Synthesis of Poly(L-lactide)/PEG Block Copolymers

PLLA(3K)/PEG(2K) diblock copolymer was prepared by a conventional methodknown in the art. S. K. Han, K. Na, Y. H. Bae, Sulfonamide basedpH-sensitive polymeric micelles: physicochemical characteristics andph-dependent aggregation, Colloids. Surf. A. Physicochem. Eng. Aspects00 (2002) 1-11.

Example 11 Synthesis of poly(L-histidine)/PEG-folate

The carboxyl group of folic acid (3 mmol) dissolved in 50 mL DMSO waspreactivated with DCC (1.2 mmol) at room temperature for 4 hours.Poly(L-N^(im)-DNP-His)/PEG-OH (1 mmol) and DMAP (a catalyst, 0.1 mmol)were added to the reactor and reacted with preactivated folic acid atroom temperature overnight. After reaction, unreacted folic acid wasremoved by dialysis (MWCO 2,000). A yellow powder was obtained afterfreeze drying. Finally, poly(N^(im)-DNP-His)/PEG-folate was thendeprotected by thiolysis with 2-mercaptoethanol to yield polyHis(5K)/PEG (2K)-folate. The yield of this conjugate was 92%.

EXAMPLE 12 Synthesis of poly(L-lactic acid)/PEG-folate

For amination, folic acid (1 mmol) dissolved in 30 mL DMSO was reactedwith DCC (1.2 mmol) and NHS (2 mmol) at 50° C. for 6 hours. Theresulting folate-NHS was mixed with ethylene diamine (10 mmol) plus 500mg pyridine and allowed to react at room temperature overnight. Thereaction was confirmed by TLC analysis (silica gel plate,2-propanol/chloroform=70/30 vol %). The crude product was precipitatedby addition of excess acetonitrile, filtered and washed three times withdiethyl ether before drying under vacuum. For further purification, thisproduct was dissolved in 2 N HCl and precipitated by adding excess8-fold acetonitrile. After filtration, the fine dark yellow powder wasdried in vacuo. The unreacted folic acid and diaminated folic acid(folate-(NH₂)₂) were separated by ion exchange chromatography. Thecolumn (10×100 mm) was packed by swollen DEAE Sephadex A-25 in 0.5 Mpotassium tetraborate solution. After the product (0.5 mg) dissolved indeionized water (20 mL) was loaded into the column, the linear ionicgradient of ammonium bicarbonate solution (10 to 30 mM) was applied. Thefolate-NH₂ solution was fractionally collected after continuous TLCanalysis as mentioned above. The folate-NH₂ solution was evaporated, andthen added NaCl to remove remaining ammonium bicarbonate. The finalproduct was obtained from recrystallization (water/acetonitrile) andvacuum drying (yield=36%). PLLA/PEG-COOH (1 mmol) was activated usingDCC (1.2 mmol) plus NHS (2 mmol) in methylene chloride. Dicyclohexylurea(DCU) was removed by filtration and excess diethyl ether added to thecrude solution, and then PLLA/PEG-NHS was obtained fromrecrystallization. The activated PLLA/PEG-NHS (1 mmol) and aminatedfolate (2 mmol) were reacted in DMSO at room temperature for two days.The unreacted amine-folate was removed by dialysis (MWCO 2,000). Thefinal yellowish product was obtained by freeze-drying. The yield of thisconjugate was 91%.

EXAMPLE 13 Preparation of pH-sensitive Mixed Micelles

The pH-sensitive micelles were prepared with polyHis/PEG with or withoutfolate and PLLA/PEG with or without folate. Before adriamycin (ADR)loading into the polymeric micelle, adriamycin hydrochloride (ADR-HCL)was stirred with 2 mole ratio of triethylamine in DMSO overnight. TheADR base (10 mg) with blended block copolymers (50 mg) at differentweight ratios (100/0, 95/5, 90/10, 75/25, 60/40, and 0/100 wt % ofpolyHis/PEG to PLLA/PEG) were dissolved in 20 mL DMSO, transferred to apreswollen dialysis membrane (Spectra/Por molecular weight cut off15,000) and dialyzed against HCl—Na₂B₄O₇ buffer solution (pH 9.0) for 24hours at 4° C. The medium was exchanged several times and the contentinside the dialysis tube was subsequently lyophilized. The amount ofentrapped ADR was determined by measuring the UV absorbance at 481 nm ofthe drug-loaded polymeric micelles dissolved in DMSO.

FIG. 12 shows the pH-dependent stability of the mixed micelles monitoredby relative light transmittance (T/T_(i)%; the transmittance of themixed micelle at each pH/transmittance at pH 9.0). The T/T_(i)% ofpolyHis/PEG micelle slightly increased with decreasing pH, especiallybelow pH 7.6, which is attributed to micelle destabilization followed bydissociation. PLLA/PEG micelle, being devoid of pH dependency, showed nochange in turbidity and preserved its stability in the entire pH rangetested. It is, however, interesting to note that the T/T_(i)% of themixed micelles rather decreased with decreasing pH. This observationseems to be associated with ionization of polyHis/PEG, accompanied byseparation and isolation of PLLA/PEG from the micelles, which in turnbecame segregated in water. This observation indirectly reflects thedisintegration of the micelles by ionization of imidazole group alongpolyHis chains. The destabilizing pH was influenced by the amount ofPLLA/PEG block copolymer in the mixed micelles. The addition of up to 10wt % of PLLA/PEG to the micelle showed a slight shift in destabilizingpH. However, 25 wt % additions considerably enhanced the micellestability at pH 7.4 and the destabilization occurred below pH 7.0. At 40wt % PLLA/PEG blend, the destabilization pH was shifted a bit furtherdownward with less release of PLLA/PEG albeit of its high content.

EXAMPLE 14 pH-dependent Adriamycin Release and Cytotoxicity

The pH-dependent micelle property prompted testing of the micelles forpH-induced drug release. When adriamycin was incorporated into the mixedmicelles (0, 10, 25 and 40 wt % PLLA/PEG) during diafiltration, themicelle sizes ranged from 50 to 80 nm as determined by dynamic lightscattering, as performed according to the procedure set out above. Thedrug loading efficacy was about 75-85% and the drug content in themicelles was 15-17 wt %.

The ADR loaded polymeric micelles were dispersed in 1 mL of phosphatebuffer saline at different pHs. The solutions were transferred in adialysis membrane tube (Spectra/Por MWCO 5,000), and the membrane wasimmersed in a vial containing 10 mL phosphate buffer saline solution atdifferent pHs. The release of ADR from micelles was tested under themechanical shaking (100 rpm) at 37° C. The outer phase of dialysismembrane was withdrawn and replaced with fresh buffer solution atpredetermined time intervals to maintain sink conditions. Themeasurement of ADR concentration by a UV/VIS spectrophotometer wasperformed after adjusting each solution pH to 8.0 using 0.1 N NaOHsolution.

FIG. 13A shows the cumulative ADR release from polyHis/PEG micelles. TheADR release pattern followed nearly first-order kinetics and reached aplateau in 24 hours and 30 wt % of loaded ADR was released for 24 hoursat pH 8.0. The release rate was accelerated by decreasing pH. ThepH-dependent release patterns of the mixed micelles are presented inFIG. 13B by plotting cumulative amount of ADR for 24 hours vs. releasemedium pH. The mixed micelles containing 25 wt % PLLA/PEG showed adesirable pH-dependency such that 32 wt % of ADR was released at pH 7.0and 70 wt % of ADR at pH 6.8 and 82 wt % at pH 5.0. On the other hand,the mixed micelles containing 40 wt % of PLLA/PEG suppressed the releaseof ADR to 35 wt % at pH 6.8 but 64 wt % at pH 6.6. This indicates thatthe content of PLLA/PEG controlled pH-dependent release from the mixedmicelles by destabilization and the transition pH coincided with theresults shown in FIG. 12. These results support the idea that the mixedmicelles truly recognize the minute difference in pH and discriminatethe tumor pH by destabilization and release rate.

Human breast adenocarcinoma (MCF-7) cells were obtained from Korean CellLine Bank (KCLB). They were maintained in RPMI-1640 medium with 2 mML-glutamine, 5% penicillin-streptomycin, 10% fetal bovine serum in ahumidified incubator at 37° C. and 5% CO₂ atmosphere.

The cells (5×10⁴ cells/mL) growing as a monolayer were harvested by0.25% (w/v) trypsin-0.03% (w/v) EDTA solution. The cells in 200 mL ofRPMI 1640 medium were seeded in a 96-well plate for 24 hours beforetest. Free ADR or ADR-loaded micelle in HClNa₂B₄O₇ buffer solution (ADR5 mg/mL, pH 9.0) was filtered through 0.2 mm syringe filters to make astock solution. After measuring ADR concentration, the solution wasdiluted to make ADR or the micelle solutions with various ADRconcentrations (100× the final concentrations (1, 10, 100, 1000, 5000and 10000 ng/mL)) used for cell cytotoxicity tests. Each solution (10μL) was diluted again with 990 μL of RPMI 1640 cell culture mediumprepared from phosphate buffered saline solution. The pH of the culturemedium containing free ADR or ADR loaded micelle was adjusted with 0.1 NHCl, or 0.1 N NaOH at a desired pH in the pH range of 6.6-8.0 prior touse. No significant pH drift in the culture medium was observedespecially when ADR concentrations above 100 ng/mL during 48 hours. Whena minor pH change was observed after 24 hours, the culture medium wascollected from the cell culture plate and its pH was adjusted with 0.01N HCl or 0.01 N NaOH. After 48 hours incubation at varying pH and ADRconcentration, the cells were washed three times with phosphate buffersaline (pH 7.4). For cytotoxicity tests of blank micelles against MCF-7cells, blank micelle concentrations (0.01, 0.1, 1, 10, 50 and 100 μg/mL)with different pHs (pH 6.6-7.4) in RPMI 1640 medium were prepared asdescribed above, in the absence of ADR. Chemosensitivity was assessedusing the tetrazolium salt MTT assay to measure the viability of tumorcells. 100 μL of medium containing 20 μL of MTT solution was added toeach well, and the plate was incubated for an additional 4 hours, andthen 100 μL of dimethylsulfoxide (DMSO) was added to each well. Thesolution was vigorously mixed to dissolve the reacted dye. Theabsorbance of each well was read using a microplate reader using a testwavelength of 570 nm and a reference wavelength of 630 nm.

When the blank micelles were tested with human breast adenocarcinoma(MCF-7) cells, no cytotoxicity was observed up to 100 μg/mL of polymericmicelle for 48 hours culture (FIG. 14) regardless of the culture mediumpH. However, ADR-loaded micelles presented tumor cell killing activityin a pH-dependent manner.

ADR loaded polyHis/PEG micelles demonstrated a certain degree of cellkilling effect at pH 7.4 because of a certain degree of micelleinstability and ADR release. But the cell viability was much reduced atpH 6.8 under the influence of enhanced ADR release (FIG. 15A). The mixedmicelles with 10 wt % PLLA/PEG (FIG. 15B) caused less cytotoxicity at pH7.4 and high cell killing effect below pH 7.2. The above results aredistinguished from the cytotoxicity of free ADR which is almostindependent of pH. The 25 wt % PLLA/PEG micelle (FIG. 15C) appeared moresensitive to pH, especially differentiating between pH 7.0 and 6.8. 40wt % PLLA/PEG micelle (FIG. 15D) distinguished pH 6.8 and 6.6 incytotoxicity. Considering that 100 wt % PLLA/PEG micelle (FIG. 15E) hadno pH-dependent cytotoxicity and no cytotoxicity of blank micelles, thepH-dependent cytotoxicity solely relies on the release of ADR atdifferent pH. The change in pH from 8.0 to 6.6 may influence cellsurface charges and cellular physiology and viability, noting that thelow pH is a favorable environment for tumor cells but opposite effect onnormal cells. The cell viability expressed in this study is relative tothose at different pH in the absence of ADR and the direct pH effect onthe cell viability was not monitored with MCF-7 cell line. No noticeabledifference in cell viability by pH with free ADR was observed.

EXAMPLE 15 Folate-Conjugated Mixed Micelles

Endogenous folic acid, a vitamin B essential for cell life, has beenutilized for tumor targeting of anticancer drugs, radiopharmaceuticals,S. D. Weitmann et al., supra; J. F. Ross et al., supra; P. S. Low & R.J. Lee, supra; J. A. Reddy & P. S. Low, supra, and genes via folatereceptor-mediated endocytosis. The receptors are highly expressed onvarious tumors such as ovarian, lung, breast, brain, colon, and kidneycancers. S. D. Weitmann et al., supra; J. F. Ross et al., supra. Afterbeing internalized, the carriers exist mainly in endosomes ormultivesicular bodies (MVD) and less in lysosomes. J. A. Reddy & P. S.Low, supra. The pH-sensitive liposomes with anticipated fusogenicactivity therefore have been utilized for drug release out of endosomesO. V. Gerasimov et al., supra; O. Meyer et al., Copolymers ofN-isopropylacrylamide can trigger pH sensitivity to stable liposomes,421 FEBS Lett. 61-64 (1998); P. S. Low & R. J. Lee, supra. Indeed, ADRentrapped in a pH-sensitive liposome-PEG that decorated with folate,enhanced cytotoxicity against tumor cells by a maximum of 86-foldcompared to free ADR due to high uptake rate of the liposomes even for ashort period of incubation time (2 hrs). P. S. Low & R. J. Lee, supra.

For active internalization of the micelles, folic acid was introducedinto the pH-sensitive mixed micelle. After internalization, the micellarcarrier actions can be combined with pH-triggered ADR release at earlyendosomal pH and the fusogenic activity of polyHis, which helps ADRrelease from endosomal compartment to cytosol. PolyHis has been reportedfor its fusogenic activity, J. M. Benns et al., supra; D. Putnam et al.,supra; C. Y. Wang & L. Huang, supra, but the mechanism of its fusogenicactivity remains unclear, and has been hypothesized with (i) theproton-sponge effect of polyHis due to protonation of imidazole atslightly acidic pH, J. M. Benns, supra, and (ii) charge-chargeinteraction, C. Y. Wang & L. Huang, supra, between endosomal membraneand poly(L-histidine).

FIGS. 16A-B demonstrate such combined effects. The ADR loadedpolyHis/PEG-folate micelles induced greatly enhanced cytotoxicity (cellviability 40% at ADR 10 μg/mL at pH 8.0 (this pH was employed to ensurethe micelle stability before internalization)), while thePLLA/PEG-folate show a slight increase in cytotoxicity. This is wellcontrasted with that of without folate conjugation where was nonoticeable difference in cytotoxicity (cell viability 80% at ADR 10μg/mL) of these two micelles at pH 8.0. These observations clarify therole of polyHis in anticancer activity by destabilization for drugrelease and fusogenicity. Besides, it was found that the mixed micellecontaining 25 wt % PLLA/PEG was more efficacious (cell viability 16% atADR 10 μg/mL).

FIG. 16B indicates that, once internalization by folate receptors grewpredominant, the pH effect on drug release became insignificant. Thisobservation can be explained by the fact that (i) the mixed micelleswere internalized into tumor cells via folate-receptor mediatedendocytosis and underwent destabilization in slightly acidic earlyendosomal compartments by protonation of imidazole group and (ii) ADRreleased from the mixed micelles under influence of extracellular pH,regardless of folate-receptor mediated endocytosis, is also effectivedrug portion for cell killing (FIGS. 15A-E). It is thought that becausethe extracellular pH in a solid tumor is heterogeneous with adistribution as a function of distance from capillary, G. R. Martin & R.K. Jain, Noninvasive measurement of interstitial pH profiles in normaland neoplastic tissue using fluorescence ratio imaging microscopy, 54Cancer Res. 5670-5674 (1994), the mixed mechanisms of triggered releasein the extracellular space and/or for cytosolic delivery afterinternalization in the in vivo situation may occur. This is expected tobe beneficial to treat solid tumors by minimizing the “road block”effect of the particles after extravasation due to the micelledestabilization and internalization into the tumor cells, increasinglocal concentration for drug diffusion to remote tumor cells as well aspassive diffusional internalization in conjunction with more activeinternalization.

The effect of PLLA/PEG in the ADR-loaded mixed micelle against MCF-7cells was apparent in FIG. 16B, although its mechanism was not clear, ascompared to the cytotoxicity of polyHis/PEG micelles tested at pH 8.0 toendow the micelle stability before internalization. The 25 wt %PLLA/PEG-folate in ADR-loaded mixed micelles showed 16-20% cellviability by ADR released in or out of cells. The micelle with 40 wt %PLLA/PEG-folate was attributed to slightly decreased cytotoxicity (cellviability 34% at ADR 10 μg/mL) and this probably was due to decreasedpH-sensitivity and slower release kinetics of ADR even at low pH.

FIG. 17 shows the cell killing rates of the mixed micelles and free ADR.At pH 6.8, the ADR-loaded mixed micelle may exhibit two separatedmechanisms of drug availability to the cells as mentioned before, i) theADR release inside of cell after internalization and (ii) outside ofcell via pH-responsive ADR release. However, free ADR diffuses only tocells passively. Unlike free ADR, therefore, folate-conjugated mixedmicelles facilitated faster cell killing in 0-4 hrs incubation andconfined to slow cell killing rate at 4-24 hrs. This observation isconsistent with the high affinity of folate-receptors on tumor cells. Inaddition, there is little difference in cell killing rate between pH 6.8and pH 7.4 (data not shown), thus explaining quick cytotoxic action ofADR-loaded mixed micelles against MCF-7 cells due to the activeinternalization. P. S. Low & R. J. Lee, supra; J. A. Reddy & P. S. Low,supra.

To visualize the effect of folate-mediated endocytosis and fusogenicactivity of the mixed micelles, the distribution of ADR on MCF-7 cellswas observed by confocal microscopy (FIGS. 18A-C). The intracellulardistribution of ADR was observed with the cells grown on a Lab-Tek^(R)II chamber slide (Nalge Nunc International, Naperville, Ill.). The RPMI1640 medium (pH 6.8) containing free ADR or ADR-loaded micelle for cellculture was prepared as described above. The ADR concentration used was1000 ng/mL in free ADR solution or micelle solutions. The treated cellswith the micelles or free ADR were washed three times with phosphatebuffer saline (pH 7.4) aqueous solution after 1 hr incubation. The cellswere fixed with 1% formaldehyde in phosphate buffer saline for 10 min atroom temperature. A coverslip was mounted on a glass microscope slidewith a drop of anti-fade mounting media (5% n-propyl galate, 47.5%glycerol and 47.5% Tris-HCl, pH 8.4) to reduce fluorescence photobleaching. ADR distribution was examined by confocal microscopy (LeicaTCS NT, Leica, Germany) at excitation and emission wavelengths of 488 nmand 510 nm, respectively. By one hour incubation time, thefolate-conjugated micelles were rapidly taken up by the cells. Thefolate-conjugated mixed micelle and PLLA/PEG-folate micelles showed highintracellular ADR concentration, which was visualized by red-intensityof ADR. Nevertheless, in the case of PLLA/PEG-folate micelles (FIG.18B), ADR significantly localized probably in endosomes instead of broaddistribution of ADR in cytosolic compartment. This observation isclearly distinguished from that of the folate-conjugated mixed micellewith fusogenic activity. ADR carried by the mixed micelles strikinglypromoted cytosolic distribution of ADR (FIG. 18C). In contrast, for freeADR (FIG. 18A), only low red-intensity appeared in the peripheral regionof the cells due to slow diffusion process into the cells for one hourincubation period.

PolyHis/PEG (or polyHis/PEG-folate) was constituted to novelpH-sensitive polymeric mixed micelles with PLLA/PEG (orPLLA/PEG-folate). This blending shifted ADR triggering pH from 7.4 to7.2-6.6. In in vitro the cell viability study, it was found that theADR-loaded mixed micelles were advantageous for tumor cell killingbecause the triggering pH for ADR release was around tumor pH_(e) (pH7.2-6.6) and there was minimal cytotoxicity at pH 7.4. Furthermore, theintroduction of folate into mixed micelles enhanced the cell killingeffect by active internalization. The fusogenic activity of polyHis inendosomes facilitated cytosolic delivery of ADR and explained theimproved cytotoxicity of the micelles to tumor cells. The combinedmechanisms of pH-triggered release and active internalization can bebeneficial to treat solid tumors by minimizing the “road block” effectof the particles after extravasation, increasing local concentration fordrug diffusion and more active internalization, but this hypothesisrequires further investigation for proof.

Adriamycin Release from Endosomal Compartments

A major limitation of chemotherapy is the multidrug resistance (mdr)phenotype of tumor cells. Although numerous studies on the reversal ofmdr have been done, M. M. Gottesman & I. Pastan, 62 Ann. Rev. Biochem.385-427 (1993); F. Thiebaut et al., 84 Proc. Nat'l Acad. Sci. USA7735-7738 (1987); N. Baldini et al., 68 Eur. J. Cell Biol. 226-239(1995); M. M. Gottesman et al., 6 Curr. Opin. Genet. Dev. 610-617(1996); S. Dey et al., 97 Proc. Nat'l Acad. Sci. USA 10594-10599 (1997,the treatment of mdr still remains unsolved.

One of the major mdr factors, P-glycoprotein (pgp), which is encoded inhumans by the MDR1 gene and is a member of the evolutionary highlyconserved family of the ATP-binding cassette transporters, F. Thiebautet al., supra; N. Baldini et al., supra, is over-expressed on the plasmamembrane of various tumor cells, which plays an important role bypumping antitumor drug out of tumor cells. As an energy-dependentdrug-efflux pump, it lowers intercellular drug concentration belowcytotoxic threshold by extruding antitumor drug from the cells. M. Koolet al., 57 Cancer Res. 3537-3547 (1997); S. P. C. Cole et al., 258Science 1650-1654 (1992).

Therefore, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer bounddoxorubicin (DOX), J. Kopecek et al., 50 Eur. J. Pharm. Biopharm. 61-81(2000), polyalkylcyanoacrylate nanoparticles, C. E. Soma et al., 21Biomaterials 1-7 (2000), liposomal drug formulations, Y. L. Lo et al.,21 Anticancer Res. 445-450 (2001), and others have been suggested anddeveloped for overcoming P-glycoprotein.

Recently, the doxorubicin-PLURONIC® block copolymers formulation(SP1049C), A. V. Kabanov et al., 82 J. Contr. Release 189-212 (2002),showed inhibition of tumor growth 3.46-times greater than freedoxorubicin against BDF1 mice bearing resistant P388/DOX cells and 7.46times greater against BALB/c nude mice bearing resistant MCF-7/DOX^(R)cells. It was reported that the PLURONIC® block copolymers generallyinhibit P-glycoprotein activity through an ATP-depletion mechanism, A.V. Kabanov et al., supra, and result in the higher energy consumption inmdr cells.

Furthermore, it was hypothesized that a folate-PEG-liposomal carrier maybypass P-glycoprotein through nonlysosomal route like folatereceptor-mediated endocytosis, D. Goren et al., 6 Clinical Cancer Res.1949-1957 (2000), albeit relatively poor results were obtained in invitro cell experiments due to the stability of the liposomes at pH5.0-6.0 and limited fusogenic activity.

Considering the activity of polymers in intracellular compartments andthe specificity of targeting, the pH-sensitive mixed micelles describedherein were examined for the likelihood that the targeted micelles aretaken up by tumor cells with overexpressed folate receptor (FR), J. A.Reddy & P. S. Low, 15 Crit. Rev. Ther. Drug Carrier Syst. 587-627(1998); C. Pichon et al., 53 Adv. Drug Deliv. Rev. 75-94 (2001), anddisrupted simultaneously under endosomal pH influence, thus finallypresenting drug release from endocytosed micelles. The known fusogenicactivity and amphoteric property of polyHis, J. M. Benns et al., supra;D. Putnam et al., supra; A. Patchornik et al., supra, support cytosolicdrug delivery. Consequently, it was investigated whether polyHis-basedmixed micelles avoid or overcome P-glycoprotein.

EXAMPLE 16

Poly(L-histidine) (polyHis, MW 5K)/PEG(MW 2K)-folate (FIG. 19A) and PLLA(MW 3K)/PEG (MW 2K)-folate (FIG. 19B) block copolymer were prepared forconstructing pH-sensitive mixed micelles as described above. Beforeloading doxorubicin to the mixed micelle, doxorubicin-HCl (DOX-HCl,Sigma Chemical) was stirred with 2 mole ratio of triethylamine (SigmaChemical) in DMSO (J. T. Baker) overnight. The DOX base (10 mg) withblended block copolymers (50 mg, 75/25 wt % of polyHis/PEG-folate toPLLA/PEG-folate) or PLLA/PEG-folate (50 mg) or polyHis/PEG-folatecopolymer (50 mg) were dissolved in 20 mL DMSO and transferred to apre-swollen dialysis membrane (Spectra/Por molecular weight cut off15,000) and then dialyzed against NaOH—Na₂B₄O₇ (Sigma Chemical) buffersolution (pH 9.0) for 24 hours at 4° C. The medium was exchanged severaltimes and the contents inside the dialysis tube were subsequentlylyophilized. The amount of entrapped DOX was determined by measuring theUV absorbance at 481 nm of the drug loaded polymeric micelles dissolvedin DMSO. The drug loading efficacy was about 75-85 wt % and consequentlythe DOX amount in micelles based on pH-sensitive micelles (PHSM) was0.15-0.17/1.0 (DOX/polymer) ratio.

DOX-resistant MCF-7 cells (MCF-7/DOX^(R)) were selected from MCF-7 cells(Korean Cell Line Bank (KCLB)) that were stepwise exposed at 0.001-10mg/mL of free DOX. Here, cells were maintained in RPMI-1640 (Gibco,Uxbridge, UK) medium with 2 mM L-glutamine, 5% penicillin-streptomycin,and 10% fetal bovine serum in a humidified incubator 37° C. and 5% CO₂atmosphere.

To test whether or not P-glycoprotein was involved in the DOX resistantphenotype MCF-7 (MCF-7/DOX^(R)) subline, the resistant MCF-7/DOX^(R) orwild MCF-7 cells (1×10⁶ cells) suspended in 200 mL phosphate buffersolution (PBS pH 7.4) containing 0.1% NaN₃ and 1% bovine serum albumin(Sigma) were incubated for 30 min at 4° C. with MRK-16 (Kamiya, Seattle,Wash.). After washing cells with ice-cold phosphate buffer solutioncontaining 0.1% NaN₃ (Sigma), 1% bovine serum albumin, and 0.002% EDTA(Sigma), cells were centrifuged to remove unbound antibody andre-suspended in fresh PBS containing 0.1% NaN₃ and 1% bovine serumalbumin. The cells were again incubated for 30 min at 4° C. with goatanti-mouse IgG fluorescein-conjugate (Sigma) used at a working dilutionof 1:50. After washing, the cells were immediately analyzed on a flowcytometer (Becton Dickinson FacScan). As negative controls, MCF-7 orMCF-7/DOX^(R) cells were incubated with only secondary antibody (goatanti-mouse IgG fluorescein-conjugate) for 30 min at 4° C. Thefluorescence signal of cells treated only with secondary antibody werecompared. A. Molinari et al., 59 Int. J. Cancer 789-795 (1994). For thecytotoxicity test of free DOX or drug-loaded polymeric micelles againstsensitive MCF-7 or MCF-7/DOX^(R) cells, cells (5×10⁴ cells/mL) growingas a monolayer, were seeded at 96-well dish in 200 mL of RPMI 1640medium before 24 hrs. Free DOX or DOX loaded micelles in RPMI 1640medium with pH 7.4 or 6.8 were prepared immediately before use asdescribed above, added to medium-removed 96-well dish with different DOXconcentration, and incubated for 48 hrs. Chemosensitivity was assessedusing the tetrazolium salt MTT (Sigma Chemical Co.). Then, 100 mL of PBSpH 7.4 containing 20 mL of MTT (500 mg/L) solution was added to eachwell. The plates were incubated for an additional 4 hrs, and then 100 mLof DMSO was added to each well. The absorbance of each well was read ona microplate reader using a test wavelength of 570 nm and a referencewavelength 630 nm.

The analysis of intracellular distribution of DOX was carried out onMCF-7 or MCF-7/DOX^(R) cells grown on a Lab-TekR II chamber slide (NalgeNunc International, Naperville, Ill.). The DOX content in micelles wasadjusted to be equivalent to free DOX (1 mg/mL). The drug-treated cellsat pH 7.4 for 1 hr incubation were washed three times with PBS pH 7.4and then cells were fixed with 1% formaldehyde (J. T. Baker) in PBS for10 min. A coverslip was mounted on a glass microscope slide with a dropof anti-fade mounting media (5% n-propyl galate, 47.5% glycerol and47.5% Tris-HCl pH 8.4, Gibco Co., Uxbridge, UK). All specimens for thedetection of DOX were examined under a confocal microscopy (Leica TCSNT, Leica, Germany) and the excitation and emission wavelengths were 488nm and 510 nm, respectively.

FIG. 20A shows the flow cytometric determination of P-glycoprotein onMCF-7/DOX^(R) cells. The MRK-16 antibody specifically recognizes andbinds to P-glycoprotein. A. Molinari et al., supra. The MRK-16 bindingto P-glycoprotein was significantly detected in the MCF-7/DOX^(R) cells.The MCF-7/DOX^(R) showed a median fluorescence peak at approximately218.5 compared to 6.23 for the sensitive MCF-7 cells. Here, thedifference of fluorescence signal between MCF-7 and MCF-7/DOX^(R) cellstreated with only secondary antibody only was negligible. This resultmeans that MCF-7/DOX^(R) cells over-expressed P-glycoprotein by chronicexposure to DOX. In addition, FIG. 20B indicates the decreased cytotoxicaction of free DOX against MCF-7/DOX^(R) cells due to theover-expression of P-glycoprotein.

FIGS. 21A-B revealed a different distribution of DOX in MCF-7 cells(FIG. 21A) or MCF-7/DOX^(R) (FIG. 21B). There was little DOX incytosolic compartment resulting from response of P-glycoprotein,DOX-efflux pump, as shown in FIG. 21B. Unlike MCF-7/DOX^(R) cells,DOX-sensitive MCF-7 cells accumulated much DOX in cytosolic compartmentby passive diffusion of DOX (FIG. 21A). This visual observation suggeststhe effective DOX-efflux pump which blocks internalization of DOX andsecretes DOX outside of cells.

FIGS. 22A-C show the effect of free DOX or DOX-loaded micelles againstMCF-7/DOX^(R) cells. The viability of MCF-7/DOX^(R) cells is littlereduced at even high concentration of free DOX, which is corresponds todrug-efflux function of P-glycoprotein. Furthermore, even if it ishypothesized that folate receptor-mediated endocytosis route is notaffected by P-glycoprotein, DOX-loaded PLLA/PEG-folate micelles do notshow significant cytotoxicity against MCF-7/DOX^(R) cells. This is muchdifferent from that DOX-loaded polyHis/PEG-folate micelles enhancedcytotoxicity. The proton-sponge effect of polyHis has been referred toas fusogenic activity because the charged polyHis reacts with theendosomal membrane. This fusogenic activity of polyHis exhibited adistinct difference as compared to PLLA/PEG-folate micelles, which isattributed to the avoiding of drug sequestration mechanism of mdr andthe endosomal disruption by polyHis. In addition, FIG. 22A alsoindicates an interesting phenomenon in that the pH-sensitive micellesdemonstrate improved cytotoxicity against MCF-7/DOX^(R) cells thanpolyHis/PEG-folate micelle. There is presumably additional fusogenicactivity, such as a certain reaction between PLLA/PEG-folate releasedfrom mixed micelle after destabilization in endosomal pH and endosomalmembrane. This is consistent with results at pH 7.4 (FIG. 22B). Therelevant mdr cell killing by PHSM is detected at pH 7.4, even if thecytotoxicity of DOX-loaded polyHis/PEG-folate micelles is reduced instudies because of relatively instability at pH 7.4.

FIG. 22C shows the feasibility of mdr reversal by pH-sensitive micellesat tumor extracellular pH, including that efficacy of mixed micelles atpH 6.8 was reduced a little bit because such micelles begins to bedestabilized below pH 7.0 and DOX released outside the cells is noteffective for killing MCF-7/DOX^(R) cells. From this study, it isapparent that after folate receptor-mediated endocytosis, fusogenicactivity of polymers released from disrupted micelles is responsible formdr reversal of MCF-7/DOX^(R) cells. Furthermore, folate receptor withhigher affinity pH-sensitive micelles in an extent and contributes tomdr reversal of MCF-7/DOX^(R) cells through endocytosis pathway even atpH 6.8. These cell viability results strongly support the feasibilitythat L-histidine based polymeric micelles can bypass the drug pumpingmechanism of mdr cells via folate receptor mediated endocytosis.

For further characterizing fusogenic activity of pH-sensitive micelles,confocal microscopy studies were done to verify distribution of DOX incells. FIG. 23B shows the localized DOX distribution in MCF-7/DOX^(R)cells treated with DOX-loaded PLLA/PEG-folate micelle at pH 6.8 in RPMI1640 medium. The entrapped PLLA/PEG-folate micelles in endosome ormultivesicular bodies (MVD) are responsible for the discrete punctuateDOX intensity and biased DOX intensity distribution. Such is conductedby the consequence of due to the absence of fusogenic activity. UnlikePLLA/PEG-folate micelle, pH-sensitive micelles (FIG. 23A) lead to, notonly broad spreading of DOX in cytosolic compartments, but alsoincreasing of DOX concentration inside of cells. As shown in FIGS.22A-C, enhanced cytotoxicity of pH-sensitive micelles is again explainedwith (i) pH-dependent DOX release behavior and (ii) fusogenic activityof decomposed polymers from observing broader DOX intensity in cells.

Considering that the folate receptor is highly populated on solid tumorcompared to normal cells, this drug delivery system will be effectivefor the specificity of tumor targeting for folate receptor-bearing solidtumors and treatment of tumor with mdr phenotype.

In Vivo Treatment of Warm-blooded Animals Using pH-Sensitive MixedPolymeric Micelles for Drug Delivery EXAMPLE 17 Growth Inhibition ofHuman Breast Carcinoma (MCF-7) Xenografts

A human breast carcinoma (MCF-7) cell line (ATCC, Manassas, Va.), whichis known to over-express folate receptors on cell surfaces, wasmaintained in RPMI 1640 medium (Sigma Chemical Co., St. Louis, Mo.) with2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 1 mM sodium pyruvate, 0.01mg/mL bovine insulin, 5% penicillin-streptomycin, and 10% fetal bovineserum in a humidified incubator at 37° C. and 5% CO2 atmosphere. Femalenude mice (BALB/c mice, Charles River Laboratories, Wilmington, Mass.),5-6 weeks old, were maintained in autoclaved microisolator cages housedin a positive pressure containment rack and cared under the guidelinesof an approved protocol from University of Utah Institutional AnimalCare and Use Committee.

To establish human breast cancer xenografts, a cell suspension (100 μL)containing 5×10⁶ MCF-7 cells was injected into the left inguinal mammaryline according to the procedure of P. J. Vickers et al., Amultidrug-resistant MCF-7 human breast cancer cell line which exhibitscross-resistance to antiestrogens and hormone-independent tumor growthin vivo, 10 Mol. Endocrinol. 886-892 (1988). The MCF-7 cell lineresponds to estrogen with an increased level of progesterone receptors.However, the estrogen response is lost in MCF-7/DOX^(R) cells.Therefore, MCF-7 tumor growth was facilitated by feeding 1 mg estrone(Sigma Chemical Co.) per 1 liter water to mice, according to theprocedure of V. Dubois et al., CPI-0004Na, a new extracellularlytumor-activated prodrug of doxorubicin, 62 Cancer Res. 2327-2331 (2002).The administration of estrone in water to mice bearing MCF-7 xenograftswas stopped one week before antitumor study.

Tumor volume was calculated using a formula, tumorvolume=0.52×length×width×height. Dimensions were measured with anelectronic digital caliper. Two to three weeks after inoculation (tumorvolume 150-200 mm³) in vivo antitumor studies were performed. Saline(control), free DOX, or DOX-loaded micelles in aqueous saline solutionwere injected intravenously through a tail vein by 1-3 bolus injectionsat three day intervals. The injection volume (approximately 0.1 mL/10 gbody weight) of micelle preparation was adjusted to give 10 mg DOX/kgbody weight. Five to eight mice per study were used. Tumor volume andbody weight were monitored with elapsed time. A mean standard error wascalculated for each experimental point.

For the preparation of doxorubicin-loaded micelles, doxorubicin.HCl(DOX.HCl, Sigma Chemical Co., St. Louis, Mo.) was desalted by stirringovernight in the presence of two molar equivalents of triethylamine(Sigma Chemical Co.) in DMSO (Sigma Chemical Co.). After DOXpurification and drying, DOX (2 mg) and blended copolymers (20 mg) ofpolyHis-b-PEG-folate (or polyHis-b-PEG without folate) andpLLA-b-PEG-folate (or pLLA-b-PEG without folate) at a blending ratio of75:25 by weight for pH-sensitive micelles (PHSM or PHSM/f, if conjugatedwith folate) or 0:100 for pH-insensitive micelles (PHIM or PHIM, ifconjugated with folate), were dissolved in 10 mL DMSO. The mixture wastransferred to a pre-swollen dialysis membrane (Spectra/Por molecularweight cut off 15,000) and then dialyzed against NaOH—Na₂B₄O₇ (SigmaChemical Co.) buffer solution at pH 9.0. The buffer was replaced withfresh buffer at 1, 2, 4, 6, 12 and 24 h. The amount of entrapped DOX wasdetermined by measuring UV absorbance at 481 nm of the drug afterdissolving micelles in DMSO. The drug loading efficacy was about 75-85wt % and, consequently, the amount of DOX in micelles based on theamount of blended polymer was 0.15-0.17/1.0 (DOX/polymer) weight ratio.

FIG. 24A shows the in vivo results of the anticancer activity of thetested DOX formulations injected intravenously on days 0 and 3. TheDOX-loaded PHSM (equivalent DOX=10 mg/kg) exhibited significantinhibition (P<0.05 compared with free DOX or saline solution) of thegrowth of MCF-7 xenografts. The tumor volume of mice treated with thePHSM (P<0.05 compared with free DOX) was approximately 4.5 or 3.6 timessmaller than that compared to saline solution or free DOX treatmentafter 6 weeks. The triggered release of DOX by tumor extracellular pH(pH_(e)<7.0) after accumulation of the micelle in the tumor sites viathe enhanced permeation and retention (EPR) mechanism may present a moreeffective modality in tumor chemotherapy, providing higher localconcentrations of the drug at tumor sites (targeted high-dose cancertherapy), while providing a minimal release of the drug in micellesduring circulating in the blood (pH 7.4). When compared with PHIM, thevolume of tumors treated with the PHSM was approximately 3.0 timessmaller than that treated with PHIM (particle size=50-70 nm, data nowshown) of which properties are not influenced by pH. In addition, thedestabilization of PHSM (converting to unimers) may help extravasationof additional micelles from the blood compartment and their penetrationinto tumors by reducing the physical barrier effect, which can begenerated by stable particles, such as conventional liposomes andpolymeric micelles. It was reported that drug carriers, such as PEGmodified liposomes, are in an insoluble form and only reside in thevicinity of the leaky blood vessels after extravasation, rathermigrating into the deep sites of tumors. This “road block effect” couldobstruct the additional accumulation of the carriers at tumor sites. Itis interesting to note that only PHSM administration showed decreasedtumor size for initial one week. No obvious changes in mice body weightin experimental groups except free DOX were observed. Free DOX causedmore weight loss after intravenous administration (FIG. 24B) thanmicelle formulations.

EXAMPLE 18 Growth Inhibition of Human Breast Carcinoma (MCF-7/DOX^(R))Xenografts

The MCF-7/DOX^(R) xenografts in nude mice were used for theinvestigation of in vivo efficacy according to the procedure of Example17. The tumor-bearing animals were treated by multiple intravenousinjections on days 0, 3 and 6 (FIGS. 25A-B). Complete tumor growthregression and toxicity-induced death were not observed in allexperimental groups. However, the tumor growth in mice treated by PHSM/fwas inhibited and the size was reduced for 2 weeks after the thirdinjection. The tumor volume in mice treated by PHSM/f (P<0.05 comparedwith free DOX) was approximately 2.7 times smaller than those treatedwith free DOX or PHIM and approximately 1.9 times smaller than thosetreated with PHSM after 6 weeks. These results indicate that thefolate-mediated endocytosis pathway of PHSM/f enhances the regression oftumors and is more efficient for MDR tumor chemotherapy than PHSM.However, the tumor regression efficacy in PHIM/f was not significantbecause some folate conjugates may recycle back to the surface beforedrug unloading. In addition, slow or limited drug release from PHIM mayassociate with drug sequestration in acidic organelles in MDR cells. Theactive internalization, enhanced drug release rate and endosomalescaping activity justify the efficacy of PHSM/f.

Free DOX administration exhibited more weight loss in mice than thegroups treated by any micelles (FIG. 25B), demonstrating less toxicityof DOX that was carried by micelles.

EXAMPLE 19 Growth Inhibition of Human Non-Small Lung NCI—H358 CarcinomaXenografts

In this example, the procedure of Example 17 was carried except that thecells injected into the mice were human non-small lung carcinoma(NCI-H358) cells, and in vivo antitumor studies were performed 1-2 weeks(tumor volume 40-70 mm³) after inoculation.

In vivo efficacy of PHSM/f was demonstrated with human non-small lungNCI-H358 carcinoma xenografts, which have characteristics of a fastgrowing and an intrinsic resistance to anticancer drugs. The in vivoresults are presented in FIG. 26A and clearly demonstrate that the PHSMmicelles are superior in tumor suppression as compared to PHIM and freeDOX. After a single intravenous injection (day; 0) of the PHSM/f, tumorvolume was reduced to minimal detectable size in 4 days. After 3 weeks,the tumor volume of mice treated by PHSM/f (P<0.05 compared with freeDOX) was approximately 4.3 or 3.8 times smaller than those compared tofree DOX or PHIM/f. No significant change in the weight was observed(FIG. 26B).

1. A method for treating a warm-blooded animal with a drug comprising:(a) mixing the drug with a pH-sensitive mixed polymeric micellecomposition comprising (i) poly(L-histidine)-poly(ethylene glycol) blockcopolymer and poly(L-lactic acid)-poly(ethylene glycol) block copolymer,(ii) poly(L-histidine)-poly(ethylene glycol) block copolymer-folate andpoly(L-lactic acid)-poly(ethylene glycol) block copolymer, (iii)poly(L-histidine)-poly(ethylene glycol) block copolymer andpoly(L-lactic acid)-poly(ethylene glycol) block copolymer-folate, or(iv) poly(L-histidine)-poly(ethylene glycol) block copolymer-folate andpoly(L-lactic acid)-poly(ethylene glycol) block copolymer-folate, toresult in a drug-loaded mixed micelle composition; and (b) administeringthe drug-loaded mixed micelle composition to the animal such that thedrug-loaded mixed micelle composition is systemically circulated in theanimal, wherein the drug-loaded mixed micelle composition is stable inblood and releases the drug in acidic conditions.
 2. The method of claim1 wherein the drug is hydrophobic.
 3. The method of claim 1 wherein thedrug is an anticancer drug.
 4. The method of claim 1 wherein the drugcomprises adriamycin.
 5. The method of claim 1 wherein the pH-sensitivemixed polymeric micelle composition comprises about 60-90% by weight ofpoly(L-histidine)-poly(ethylene glycol) block copolymer orpoly(L-histidine)-poly(ethylene glycol) block copolymer-folate and about10-40% by weight of poly(L-lactic acid)-poly(ethylene glycol) blockcopolymer or poly(L-lactic acid)-poly(ethylene glycol) blockcopolymer-folate.
 6. The method of claim 5 wherein the pH-sensitivemixed polymeric micelle composition comprises about 75% by weight ofpoly(L-histidine)-poly(ethylene glycol) block copolymer orpoly(L-histidine)-poly(ethylene glycol) block copolymer-folate and about25% by weight of poly(L-lactic acid)-poly(ethylene glycol) blockcopolymer or poly(L-lactic acid)-poly(ethylene glycol) blockcopolymer-folate.
 7. The method of claim 1 wherein the warm-bloodedanimal is a human.
 8. A method for treating multidrug resistance in awarm-blooded animal comprising administering into the systemiccirculation of the animal a drug-loaded mixed micelle compositioncomprising a mixture of a hydrophobic anticancer drug and pH-sensitivemixed polymeric micelles comprising (i) poly(L-histidine)-poly(ethyleneglycol) block copolymer and poly(L-lactic acid)-poly(ethylene glycol)block copolymer, (ii) poly(L-histidine)-poly(ethylene glycol) blockcopolymer-folate and poly(L-lactic acid)-poly(ethylene glycol) blockcopolymer, (iii) poly(L-histidine)-poly(ethylene glycol) block copolymerand poly(L-lactic acid)-poly(ethylene glycol) block copolymer-folate, or(iv) poly(L-histidine)-poly(ethylene glycol) block copolymer-folate andpoly(L-lactic acid)-poly(ethylene glycol) block copolymer-folate.
 9. Themethod of claim 8 wherein the hydrophobic anticancer drug comprisesadriamycin.
 10. The method of claim 8 wherein the pH-sensitive mixedpolymeric micelles comprise about 60-90% by weight ofpoly(L-histidine)-poly(ethylene glycol) block copolymer orpoly(L-histidine)-poly(ethylene glycol) block copolymer-folate and about10-40% by weight of poly(L-lactic acid)-poly(ethylene glycol) blockcopolymer.
 11. A method for treating a warm-blooded animal with a drugcomprising: (a) mixing the drug with a pH-sensitive mixed polymericmicelle composition comprising (i) poly(L-histidine)-poly(ethyleneglycol) block copolymer or poly(L-histidine)-poly(ethylene glycol) blockcopolymer-targeting moiety and (ii) an amphiphilic polymer oramphiphilic polymer-targeting moiety, to result in a drug-loaded mixedmicelle composition; and (b) administering the drug-loaded mixed micellecomposition to the animal such that the drug-loaded mixed micellecomposition is systemically circulated in the animal, wherein thedrug-loaded mixed micelle composition is stable in blood and releasesthe drug in acidic conditions.
 12. The method of claim 11 wherein thetargeting moiety is folate.
 13. The method of claim 11 wherein theamphiphilic polymer comprises poly(L-lactic acid)-poly(ethylene glycol)block copolymer.
 14. The method of claim 11 wherein the amphiphilicpolymer comprises poly(DL-lactic-co-glycolic acid).
 15. The method ofclaim 11 wherein the amphiphilic polymer comprises an ABA blockcopolymer.
 16. The method of claim 15 wherein the ABA block copolymercomprises a poly(ethylene oxide)-poly(propylene oxide)-poly(ethyleneoxide) block copolymer.
 17. A method for treating a warm-blooded animalwith a drug comprising: (a) mixing the drug with a pH-sensitive mixedpolymeric micelle composition to result in a drug-loaded mixed micellecomposition wherein the drug-loaded mixed micelle composition is stablein blood and releases the drug in acidic conditions; and (b)administering the drug-loaded mixed micelle composition to the animalsuch that the drug-loaded mixed micelle composition is systemicallycirculated in the animal.
 18. The method of claim 17 wherein thewarm-blooded animal is a human.
 19. The method of claim 17 wherein thedrug is an anticancer drug.
 20. The method of claim 17 wherein thepH-sensitive mixed polymeric micelle composition comprisespoly(L-histidine)-poly(ethylene glycol) block copolymer and anamphiphilic polymer.
 21. The method of claim 20 wherein thepoly(L-histidine)-poly(ethylene glycol) block copolymer, or theamphiphilic polymer, or both the poly(L-histidine)-poly(ethylene glycol)block copolymer and the amphiphilic polymer are covalently coupled to atargeting moiety.
 22. The method of claim 21 wherein the targetingmoiety comprises folate.
 23. The method of claim 17 wherein theamphiphilic polymer is a member selected from the group consisting ofpoly(L-lactic acid)-poly(ethylene glycol) block copolymer,poly(DL-lactic-co-glycolic acid), poly(ethylene oxide)-poly(propyleneoxide)-poly(ethylene oxide) block copolymer, and mixtures thereof.